CN110520154B

CN110520154B - Multivalent streptococcus pneumoniae vaccine compositions

Info

Publication number: CN110520154B
Application number: CN201880025013.0A
Authority: CN
Inventors: 赵成济; 崔淑英; 朴惠梶
Original assignee: LG Chem Ltd
Current assignee: LG Chem Ltd
Priority date: 2017-03-15
Filing date: 2018-03-14
Publication date: 2023-08-04
Anticipated expiration: 2038-03-14
Also published as: KR20180105590A; KR102101879B1; CN110520154A; WO2018169303A1

Abstract

The present invention relates to a streptococcus pneumoniae (Streptococcus pneumoniae) vaccine composition, more specifically a vaccine composition is provided comprising: (i) capsular polysaccharide-protein conjugates; (ii) 2-phenoxyethanol (2-PE); and (iii) formaldehyde (HCHO), and methods of preparing the vaccine compositions are provided.

Description

Multivalent streptococcus pneumoniae vaccine compositions

Technical Field

The present invention relates to vaccine compositions against pneumococci (streptococcus pneumoniae (Streptococcus pneumoniae)), more specifically to a vaccine composition comprising (i) a capsular polysaccharide-protein conjugate, (ii) 2-phenoxyethanol (2-PE) and (iii) formaldehyde (HCHO), and a method of preparing the vaccine composition.

Background

Pneumococci (streptococcus pneumoniae (Streptococcus pneumonia)) is a gram-positive and haemolytic streptococcus strain and is a major cause of meningitis, pneumonia and serious invasive infections in infants, children and elderly worldwide. Over 160 tens of thousands of people die each year from pneumococcal disease (2008, world health organization (International Health Organization) data), especially in children under 5 years old and elderly over 65 years old who have low immunity, the incidence of invasive infectious diseases caused by pneumococci is high.

Pneumococci are classified into about more than 90 serotypes according to the structure and immunological characteristics of capsular polysaccharides as major virulence factors surrounding their outer parts (cell membranes), and about 20 of them are known to be associated with 80-90% of pathogenicity in humans. The only host for pneumococci is humans, and they usually colonize the healthy normal nasopharynx (20-40% of infants, 5-10% of adults). In 2005, the center for disease prevention and control (Centers for Disease Control and Prevention) (CDC) reported that children under about 210 ten thousand years old die from pneumonia in developing countries alone annually, 120 ten thousand of them die from pneumococcal infection, and that meningitis and sepsis caused by pneumococci occur in the United states approximately 3,000 and 50,000 cases annually, respectively (Peters TR, poehling KA et al, invasive pneumococcal disease (Invasive pneumococcal disease), JAMA 2007; 297:1825-6). Furthermore, according to the pneumoACTION database as a database of pneumococcal diseases, the number of incidences of pneumococcal infection in children in korea was 24,047 per year in 2000, and 47 of them died (www.pneumoadip.org). Furthermore, according to the latest "study of serotype analysis of pneumococci in korea children and adolescents" (study on the serotype analysis of pneumococcus in children and adolescents in the country) issued by the disease prevention and control center (Centers for Disease Control and Prevention), pneumococci are the most common (43.7%) cause of invasive infections in infants between 3 months and 59 months. For pneumococci causing invasive infectious diseases worldwide, multi-drug resistant bacteria showing resistance to three or more drugs are increased in addition to penicillin, and thus the treatment of pneumococcal infectious diseases is made more difficult.

Thus, there is always a need for vaccination against pneumococci in children and the elderly at high risk of pneumococcal infectious diseases.

Multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977 in order to prevent pneumococcal infectious diseases, and these capsular polysaccharide vaccines have proven useful for preventing pneumococcal diseases in the elderly and high risk patients. However, since the immune system is less mature than in adults in infants and children, it is difficult to expect to function as a vaccine in the case of administration of only polysaccharide vaccines, because of the immune systemPolysaccharide antigens cannot be recognized as external invasive factors. In order to solve such a problem that polysaccharide vaccines reduce immunogenicity in infants and children, etc., 7-valent pneumococcal conjugate vaccines (7 vPnC,) This is a capsular polysaccharide-protein conjugate vaccine that couples an immunogenicity enhancing carrier protein to a polysaccharide antigen, and has been reported in many references to be effective in preventing invasive diseases and inflammation in the middle ear in infants and children. However, the use of the 7-valent vaccine causes a reduction in invasive disease caused by the vaccine serotypes used for the vaccine, but in addition exhibits a relatively increased phenomenon of pneumococcal disease caused by some non-vaccine serotypes. Thus, 10-valent capsular polysaccharide-protein conjugate vaccine +. >And wherein->Is added with 13-valent pneumococcal conjugate vaccine of 6 serotypes +.>However, for some serotypes involved, the potential for their insufficient potency as a vaccine has been reported [ Andrews NJ et al, (2014) Lancet Infec Dis (14) 839; EMEA evaluation report of Prevenar 13 (EMEA Assessment Report for Prevenar 13), (2009) EMA/798877/2009]Thus, there is always a need for the development of new vaccine formulations that exhibit higher and more stable thresholds.

Meanwhile, the vaccine injection formulation should use a preservative to prevent contamination by microorganisms. For mixed vaccine products exported by UN et al to less developed countries, multi-dose products containing preservatives are preferred due to the country's environment, distribution method, cost, etc. Thiomerosal, 2-phenoxyethanol (2-PE), phenol, and the like are present as preservatives for vaccine products, in amounts commonly used in the art. For example, merthiolate is used at a concentration of 10 μg/mL, 2-PE is used at a concentration of 5mg/mL, and multi-dose vaccine products containing them can be commercialized if they pass the bactericidal capacity test of EP-B (european pharmacopoeia class B) or USP (united states pharmacopoeia) standards.

Thiomerthiolate is an ethyl mercury derivative compound that has been used as a preservative for multi-dose vaccine injections since the early 1930 s. Thiomerosal has been used for the purpose of preventing the growth of contaminating microorganisms and maintaining sterile conditions during storage and use of vaccine products, and many of the 5-valent liquid mixed vaccines (including D, T, P, hib, HBsAg) that have achieved WHO PQ (pre-qualification) contain such thimerosal as a preservative.

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

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

Disclosure of Invention

[ technical problem ]

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

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

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

Technical scheme

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

(i) A capsular polysaccharide-protein conjugate, wherein the capsular polysaccharide is derived from Streptococcus pneumoniae (Streptococcus pneumoniae),

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

(iii) Formaldehyde (HCHO).

In this context, a pneumococcal immunogenic composition means a composition that induces an immune response against pneumococci and is used in the same sense as a pneumococcal vaccine composition unless otherwise indicated.

The pneumococcal vaccine compositions provided herein may be immunogenic compositions with improved stability and/or antiseptic ability (bactericidal ability), wherein the efficacy (immunogenicity) of the induction of an immune response against pneumococci is maintained for a long period of time by comprising a capsular polysaccharide of streptococcus pneumoniae (Streptococcus pneumoniae) and 2-phenoxyethanol and formaldehyde as preservatives.

The capsular polysaccharide may be a capsular polysaccharide derived from streptococcus pneumoniae (Streptococcus pneumoniae). In particular, the capsular polysaccharide may be a capsular polysaccharide derived from more than 2, e.g., more than 5, more than 7, more than 9, more than 11, more than 13, or more than 14 serotypes of streptococcus pneumoniae (Streptococcus pneumoniae) serotypes. In one embodiment, the capsular polysaccharide may comprise capsular polysaccharides from 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 serotypes of streptococcus pneumoniae (Streptococcus pneumoniae) serotype. For example, the capsular polysaccharide may comprise capsular polysaccharides derived from 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 serotypes selected from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In one embodiment, the capsular polysaccharide may comprise 13 capsular polysaccharides from Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, 14 capsular polysaccharides from Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, 15 capsular polysaccharides from Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 12F, or 15 capsular polysaccharides from streptococcus pneumoniae (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 (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, but are not limited thereto.

The capsular polysaccharide-protein conjugates may be the capsular polysaccharide-protein conjugates mentioned above, each separately conjugated to a protein by conventional means, e.g. by covalent bonds, derived from more than 2 serotypes. In one embodiment, the conjugate may have a structure in which each capsular polysaccharide (specifically, the hydroxyl groups of the polysaccharide) is linked to a protein (specifically, the amino groups of the protein) via-O-C (NH) -NH-using a cyanation method ([ polysaccharide ] -O-C (NH) -NH- [ protein ]). When the capsular polysaccharide and protein are coupled using a cyanation method, it may be advantageous to maintain immunogenicity after coupling because structural modification of the capsular polysaccharide is less likely to occur than other methods (e.g., reductive amination methods, etc.). For example, it has been reported that although when some serotypes (19F, etc.) are coupled to proteins by a reductive amination method, the hexasaccharide ring structure is cleaved in accordance with the reaction combination and thus an open structure may be formed, thereby reducing immunogenicity, this problem is not likely to occur when it is coupled to proteins by a cyanation method.

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

In one embodiment, the protein may be CRM197 protein. The CRM197 protein is a non-toxic variant of diphtheria toxin, i.e. a toxoid, isolated from the medium of strain C7 of corynebacterium diphtheriae (Corynebacterium diphtheria) (CRM 197, e.g. grown in casein amino acid and yeast extract basal medium). CRM197 protein may be purified from the C7 medium of corynebacterium diphtheriae (Corynebacterium diphtheria) strain by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography, or obtained recombinantly by conventional methods, such as those disclosed in reference to U.S. Pat. No. 5,614,382 (incorporated herein by reference). In one embodiment, the CRM197 protein may comprise the amino acid sequence of GenBank accession No. 1007216A, or an amino acid sequence having 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology to the amino acid sequence.

In addition, toxoids derived from diphtheria bacteria other than the strain C7 of corynebacterium diphtheriae (Corynebacterium diphtheria) can be used as carrier proteins.

In addition, useful carrier proteins may be at least one selected from the group consisting of:

inactivated bacterial toxins, such as tetanus toxoid, pertussis toxoid, cholera toxoid (as disclosed, for example, in international patent application publication No. WO 2004/083251), exotoxins a derived from Escherichia coli (e.coli) LT, escherichia coli (Escherichia coli) ST and pseudomonas aeruginosa (Psendomonas aeruginosa);

Bacterial outer membrane proteins such as outer membrane complex C (OMPC), membrane porin, transferrin binding protein, pneumolysin, pneumococcal surface protein a (PspA), pneumococcal adhesion protein (PsaA), C5a peptidase derived from group a or group B streptococcus, haemophilus influenzae (Haemophilus influenza) protein D;

ovalbumin, keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), purified protein derivatives of tuberculin (PPD);

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

The capsular polysaccharide-protein conjugate may be a multivalent polysaccharide-protein conjugate, including conjugates in which each of the capsular polysaccharides derived from 2 or more, e.g., 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, or 14 or more serotypes of streptococcus pneumoniae (Streptococcus pneumoniae) serotypes is conjugated to a carrier protein, e.g., CRM197 protein, as mentioned above. In one embodiment, the capsular polysaccharide-protein conjugate may be a 13-24, 13-19, 13-17, 13-15, 14-24, 14-19, 14-17, or 14-15 valent polysaccharide-protein conjugate, wherein each of 13-24, 13-19, 13-17, 13-15, 14-24, 14-19, 14-17, or 14-15 serotype capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotype is conjugated to the carrier protein, e.g., CRM197 protein. For example, the capsular polysaccharide-protein conjugate may be 13-24, 13-19, 13-17, 13-15, 14-24, 14-19, 14-17 or 14-15 polysaccharide-protein conjugates, wherein each capsular polysaccharide derived from a serotype selected from streptococcus pneumoniae (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 is conjugated to the carrier protein, e.g., 197. In one embodiment, the capsular polysaccharide-protein conjugate may be: a polysaccharide-protein conjugate of valency 13 comprising each of 13 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and a carrier protein such as CRM197; a 14-valent polysaccharide-protein conjugate comprising each of the 14 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and a carrier protein such as CRM197; a 15-valent polysaccharide-protein conjugate comprising each of 15 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 12F and a carrier protein such as CRM197; a 15-valent polysaccharide-protein conjugate comprising each of the 15 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 15B and a carrier protein such as CRM197; or a 17-valent polysaccharide-protein conjugate comprising each of 17 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F and a carrier protein such as CRM197, but is not limited thereto.

The pneumococcal vaccine composition provided herein is a Pneumococcal Conjugate Vaccine (PCV) comprising the capsular polysaccharide-protein conjugate. The pneumococcal vaccine composition may be a multivalent pneumococcal vaccine composition comprising polysaccharide-protein conjugates wherein each of the capsular polysaccharides derived from 2 or more, e.g., 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, or 14 or more serotypes of streptococcus pneumoniae (Streptococcus pneumoniae) serotypes is coupled to a carrier protein, e.g., CRM197 protein, as mentioned above. In one embodiment, the pneumococcal vaccine composition may be a 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 pneumococcal vaccine composition comprising 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 polysaccharide-protein conjugates, wherein each of the 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 serovars derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes is conjugated to the carrier protein, e.g., CRM 197. For example, the pneumococcal vaccine composition may be 13-24, 13-19, 13-17, 13-15, 14-24, 14-19, 14-17 or 14-15 pneumococcal vaccine composition comprising polysaccharide-protein conjugates in which each capsular polysaccharide derived from a serotype selected from streptococcus pneumoniae (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 is conjugated to the carrier protein, e.g., CRM protein 197.

In one embodiment, the pneumococcal vaccine composition may be:

(1) A 13-valent pneumococcal vaccine composition comprising 13 polysaccharide-protein conjugates, wherein each of the 13 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are conjugated to the carrier protein, e.g., CRM197 protein;

(2) A 14-valent pneumococcal vaccine composition comprising 14 polysaccharide-protein conjugates, wherein each of the 14 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F are conjugated to the carrier protein, e.g., CRM197 protein;

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

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

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

The multivalent pneumococcal vaccine composition provided herein has significantly excellent threshold values compared to conventionally developed multivalent vaccines (e.g., 13-valent vaccine), such as 13-valent to 24-valent, 13-valent to 19-valent, 13-valent to 17-valent, 13-valent to 15-valent, 14-valent to 24-valent, 14-valent to 19-valent, 14-valent to 17-valent, or 14-valent to 15-valent pneumococcal vaccine of the above composition, and can be expected to have very excellent effects of preventing and/or treating pneumococcal infectious diseases.

The pneumococcal vaccine composition may be a single dose composition formulated for single administration or a multi-dose composition for multiple administrations. As used herein, "multi-dose" may mean a formulation unit containing a vaccine dose that may be administered (vaccinated) to 1 individual administered (vaccinated) more than once (e.g., 2 or more times) or a vaccine dose that may be administered (vaccinated) to 2 individual administered (vaccinated) more than once or more than once (e.g., 2 or more times).

In addition to pneumococcal capsular polysaccharide-protein conjugates of more than 2 serotypes, the pneumococcal vaccine composition may also include a preservative. Preservatives that may be used in the vaccine composition (e.g., injection formulation) may be at least one selected from the group consisting of 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, phenylmercuric nitrate, and the like.

In one embodiment, as an optimized form of the multivalent pneumococcal vaccine composition mentioned above, for example, the multivalent pneumococcal vaccine composition mentioned above, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15, the preservative may comprise 2-phenoxyethanol and formaldehyde. Likewise, by including 2-phenoxyethanol and formaldehyde as preservatives, the amount of 2-phenoxyethanol used is reduced, thereby reducing toxicity and/or side effects, and achieving a more improved preservative effect through the synergistic effect of the two components.

The content of 2-phenoxyethanol included in the multivalent pneumococcal vaccine composition of the invention may be below 10mg/ml, below 7mg/ml, below 6mg/ml or below 5mg/ml, e.g. above 4mg/ml and below 10mg/ml, 4mg/ml to 7mg/ml, above 4mg/ml and below 7mg/ml, 4 to 6mg/ml, 4.5mg/ml to 6mg/ml, 5mg/ml to 6mg/ml, 4 to 5mg/ml or 4.5mg/ml to 5mg/ml (as used herein, the term "to" in the numerical range refers to a range between above the lower limit and below the upper limit). The content of 2-phenoxyethanol in the vaccine composition is preferably above the lower limit of the range to exhibit a significant preservative effect, and is preferably less than or below the upper limit of the range to ensure no or acceptable toxicity and/or side effects.

The formaldehyde content included in the multivalent pneumococcal vaccine composition of the invention may be 90 to 200 μg/mL, 90 to 190 μg/mL, 90 to 180 μg/mL, 90 to 170 μg/mL, 100 to 200 μg/mL, 100 to 190 μg/mL, 100 to 180 μg/mL, or 100 to 170 μg/mL. The formaldehyde content included in the vaccine composition is preferably above the lower limit of the range to exhibit a significant preservative effect, and preferably below the upper limit of the range to ensure no or acceptable toxicity and/or side effects.

The vaccine composition is characterized by being stable for more than about 3 months, more than about 6 months, more than about 1 year, more than about 1.5 years, more than about 2 years, or more than about 2.5 years under usual storage conditions, for example, at a temperature of from 2 ℃ to 8 ℃, 20 ℃ to 25 ℃, or about 37 ℃. Herein, stability of a vaccine composition may mean that the vaccine composition maintains the original antigenicity (immunogenicity) at an equivalent level, and/or that each component is maintained without degradation or loss, and/or without infection, e.g., of bacteria/viruses, etc.

Other embodiments provide a pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infectious disease comprising the multivalent pneumococcal vaccine composition mentioned above. Other embodiments provide a method for preventing or treating pneumococcal infection or a pneumococcal infectious disease comprising administering to a subject in need of prevention or treatment of pneumococcal infection or a pneumococcal infectious disease a pharmaceutically effective dose of the multivalent pneumococcal vaccine composition mentioned above. The pneumococcal vaccine composition included in the pharmaceutical composition or the pneumococcal vaccine composition for the prevention or treatment method may be a single dose pharmaceutical composition for single administration or a multi-dose pharmaceutical composition for multiple administrations.

Pneumococcal infectious disease means all diseases caused by pneumococcal infection and may be pneumonia, inflammation in the middle ear, sinusitis, bacteremia, etc. "pneumonia" is an acute infectious disease of the lung parenchyma, its sources of infection are mainly Streptococcus pneumoniae (Streptococcus pneumoniae) and Klebsiella pneumoniae (Klebsiella pneumoniae). In particular, pneumococcal pneumonia accounts for about 50% of all pneumonia, and is a disease in which severe chills, fever, cough and chest pain occur and sputum usually bleeds, and may cause complications such as pleurisy, meningitis, endocarditis, peritonitis, and the like (diagn. Microbiol. Effect. Dis.,2001, 39:181-185).

As used herein, the term "pneumococcus" refers to Streptococcus pneumoniae (Streptococcus pneumoniae) and, in general, is a symbiotic organism that colonizes the mucosal surfaces of the human nasopharynx. When factors of the host allow the organism to approach the lower respiratory tract, a strong inflammatory response ensues and thus causes dense consolidation as the alveolar space fills with exudates, thereby causing pneumonia. Pneumococci can synthesize more than 90 structurally unique capsular polysaccharides and the serotypes of pneumococci are classified according to these structural and immunological characteristics of capsular polysaccharides. Thus, when using capsular polysaccharides of pneumococci for the preparation of a formulation for a vaccine, different immune responses may be exhibited depending on the kind of capsular polysaccharide, i.e., the serotype of pneumococci from which the capsular polysaccharide is derived.

Since the capsular polysaccharide is recognized as an antigen when administered into the body, allowing the production of antibodies thereto, it is allowed to be used to produce vaccine compositions for the prevention of pneumococci (or pneumococcal infectious diseases). In this context, the term "antigen" means a substance that can specifically elicit an immune response when invading the body. In one embodiment, 13 to 24 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, respectively, can serve as antigens.

Other embodiments provide a method for preparing a pneumococcal vaccine composition having improved stability and/or antiseptic (or bactericidal) capability, or a method for improving the stability and/or antiseptic (or bactericidal) capability of a pneumococcal vaccine. The method may include:

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

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

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

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

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

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

(i) A step of separating or separating and purifying capsular polysaccharide from Streptococcus pneumoniae (Streptococcus pneumoniae);

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

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

In other embodiments, the step (1) of preparing the capsular polysaccharide-protein conjugate may further perform one or more steps selected from the group consisting of an ultrafiltration step, a sterilization filtration step, and an adsorption step after the step of connecting the capsular polysaccharide to the protein through-O-C (NH) -NH-by performing a cyanation method. The cyanation process can be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.

In a specific example of the invention, sodium deoxycholate is used to solubilize streptococcus pneumoniae (Streptococcus pneumoniae) with 13 or 15 respectively different serotypes and release polysaccharides that are linked to the cells. Then, 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 were purified by performing a CTAB (cetyltrimethylammonium bromide) method because ionic bonds can be formed between them and CTAB, and 3 serotypes 7F, 14, and 33F that do not react with CTAB were purified using aluminum phosphate gel (Algel). The reaction with CTAB may be carried out by adding CTAB to the reactants in an amount of 0.5 to 5 wt% or 1 to 3 wt% (e.g., 2 to 3 wt% for 23F-derived polysaccharides, 1 to 2 wt% for polysaccharides derived from serotypes other than 23F) at a concentration of 1 to 20% (w/v) or 5 to 15% (w/v), but is not limited thereto. After reaction with CTAB, one or more steps of recovering the precipitate after centrifugation, re-suspending the precipitate using a sodium chloride solution (e.g., about 100 to 500 mM), and removing CTAB ions using sodium iodide may be further performed, but is not limited thereto. The aluminum phosphate gel reaction may be performed by adding an aluminum phosphate gel solution in an amount of 1 to 20 wt% or 5 to 15 wt% to the reactant, but is not limited thereto.

Since infants and children having a lower immune system than adults cannot recognize it as an antigen when using a vaccine composition using only the capsular polysaccharide, and thus an immune response may not occur, a form in which a conjugate of a carrier protein and the capsular polysaccharide is combined is prepared and used in the present invention.

"Carrier protein" refers to a protein that can enhance the immunogenicity of a capsular polysaccharide antigen by covalent binding to the polysaccharide, and the specific species are the same as described above. In one particular embodiment, CRM197 may be used. The carrier protein may be conjugated to the capsular polysaccharide by standard conjugation methods, and the capsular polysaccharide-carrier protein conjugate formed by it may be a capsular polysaccharide-carrier protein conjugate in which one or more capsular polysaccharides are conjugated to one carrier protein.

Well known methods for preparing conjugates of capsular polysaccharides and carrier proteins may be included within the scope of the present invention, and the conjugates have structures in which the capsular polysaccharide and carrier protein are linked via-O-C (NH) -NH-groups using cyanation methods. The cyanation method may be suitably performed by a person skilled in the art by a known method, for example, by using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.

As an example for preparing the capsular polysaccharide and carrier protein conjugate, the glycoconjugate may be formed by chemically activating purified capsular polysaccharides and coupling each chemically activated capsular polysaccharide to the carrier protein one after the other. Cyanation activity using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) treatment can change the hydroxyl groups of the capsular polysaccharide to cyanate groups and form covalent bonds with the amino groups of carrier protein CRM197 by using it. The cyanation reaction by CDAP can be specifically terminated by adding 3 molar equivalents of glycine solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto, and the reaction solution and reaction conditions can be appropriately controlled according to the purpose by those skilled in the art.

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

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

Herein, the term "vaccine" is an antigen-containing biological agent that provides immunity to a living body, and means an immunogen or an antigenic substance for preventing an infectious disease that generates immunity in a living body by being administered to a human or an animal.

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

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

(1) Aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.);

(2) Emulsion formulations of the water-in-oil type (with or without other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example (a) MF59 (No. wo 90/14837): containing 5% (w/v) squalene, 0.5% (w/v) tween 80 and 0.5% (w/v) Span 85 (optionally also containing various amounts of MTP-PE) and formulated into submicron particles using a microfluidizer such as a 110Y microfluidizer (Microfluidics, newton, MA), (b) SAF: containing 10% (w/v) squalene, 0.4% (w/v) Tween 80,5% (w/v) pluronic block polymer L121 and thr-MDP (see below), and forming a large particle size emulsion into a submicron emulsion or cavity by micro-jet, and (c) Ribi ^TM Adjuvant System (RAS) (cornixa, hamilton, MT): comprising one or more bacterial cell wall components from the group consisting of: 2% (w/v) squalene, 0.2% (w/v) Tween 80 and 3-O-deacylated monophosphoryl lipid A (MPL) as disclosed in U.S. Pat. No. 4,912,094 ^TM ) (Corixa), trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS), preferably MPL+CWS (Detox) ^TM )；

(3) Saponin adjuvants such as Quil A or STIMULON ^TM QS-21 (anti-genes, framingham, mass., U.S. Pat. No. 5,057,540) or particles formed therefrom (e.g., ISCOM);

(4) Bacterial lipopolysaccharide, synthetic lipid A homologs (e.g., aminoalkyl glucosamine phosphate compounds (AGPs)) or derivatives or homologs thereof (which are commercially available from Corixa and disclosed in U.S. Pat. No. 6,113,918; one example of such an AGP is 2- [ (R) -3-tetradecanoyloxy-tetradecanoylamino ] ethyl 2-deoxy-4-O-phosphono-3-O- [ (R) -3-tetradecanoyloxy-tetradecanoyl ] -2- [ (R) -3-tetradecanoyloxy-tetradecanoylamino ] -b-D-glucopyranoside, and which is also known as 529 (also previously known as RC 529), and which is formulated into an aqueous form or stable emulsion;

(5) Synthetic polynucleotides (e.g., oligonucleotides containing CpG motifs (U.S. Pat. No. 6,207,646));

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

(7) Wild-type Cholera Toxin (CT) or mutant cholera toxins, e.g. wherein the glutamic acid in amino acid position 29 is replaced by another amino acid, in particular histidine (WO-2002/098368 and WO-2002/098369), pertussis Toxin (PT) or a thermolabile toxin (LT) of Escherichia coli, in particular detoxified variants of the bacterial ADP-ribosylating toxins such as LT-K63, LT-R72, CT-S109, PTK9/G129 (WO-93/13302 and WO-92/19265); and

(8) Complement such as the trimer of complement component C3d,

but is not limited thereto.

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

The aluminium salt adjuvant may be an alum precipitated vaccine or an alum adsorbed vaccine. The aluminum salt may include alumina hydrate, alumina Trihydrate (ATH), aluminum hydrate, aluminum trihydrate, aluminum gel, syperfos, aluminum hydroxide gel, aluminum hydroxide, aluminum hydroxy phosphate (aluminum phosphate adjuvant (APA)), amorphous alumina, and the like, but is not limited thereto. APA means a suspension of aluminum hydroxy phosphate. It can be prepared as follows: aluminum chloride was mixed with sodium phosphate in a ratio of 1:1 (by volume) to precipitate aluminum hydroxy phosphate sulfate, and the precipitate was made to have a size of 2 to 8 μm, which was then dialyzed with an aqueous salt solution and sterilized. As an embodiment, use is made ofCommercially available Al (OH) ₃ (e.g., alumina gel or superfos) to adsorb proteins. 50-200 g of protein can be adsorbed per 1mg of aluminium hydroxide and the ratio depends on the pI of the protein and the pH of the solvent. Proteins with low pI bind more strongly than proteins with high pI. The aluminum salt can non-specifically activate macrophages, complement and innate immune mechanisms by forming antigen storage sites that slowly release antigen for 2-3 weeks.

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

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

In particular, when the vaccine composition of the present invention is intended for parenteral administration, the buffering agent may be selected among buffering agents suitable for USP. For example, the buffer may be at least one selected from monoacids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid, dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid, bases such as ammonia, diethanolamine, glycine, triethanolamine, TRIS.

Furthermore, the vaccine composition of the present invention may comprise a non-ionic detergent. For example, it may contain surfactants such as polysorbate 20 and polysorbate 80, in particular polyoxyethylene sorbitan esters (commonly known as tween); copolymers of Ethylene Oxide (EO), propylene Oxide (PO) and Butylene Oxide (BO) (e.g., dowfax (tm)); oxynol, in particular oxynol-9 (Triton-100), having a different number of repetitions of ethoxy (oxy-1, 2-ethanediyl) groups, respectively; ethyl phenoxy polyethoxy ethanol (IGEPAL CA-630/NP-40); nonylphenol ethoxylates such as the NP series; polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethylene glycol monolauryl ether (Brij 30); sorbitan ethers known as SPAN, in particular sorbitan trioleate (SPAN 85), but are not limited thereto.

Tween 80 may be included in the emulsion and a mixture with a non-ionic detergent such as tween 80/Span 85 may be used. A combination of polyoxyethylene sorbitan esters such as tween 80 and oxinols such as Triton X-100 is suitable and a combination of Laureth9 with tween or oxinols is also useful. In particular, from 0.01% (w/v) to 1% (w/v), in particular from 0.1% (w/v), of a polyoxyethylene sorbitan ester (such as tween 80); 0.001% (w/v) to 0.1% (w/v), particularly 0.005% (w/v) to 0.02% (w/v) of octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (e.g., triton X-100); and 0.1% (w/v) to 20% (w/v), preferably 0.1% (w/v) to 10% (w/v), especially 0.1% (w/v) to 1% (w/v) or about 0.5% (w/v) of a polyoxyethylene ether (such as laureth 9).

The vaccine compositions of the present invention may be formulated in single dose vials, multi-dose vials or pre-filled syringes. Thus, one embodiment provides a vial or prefilled syringe containing a single dose or multiple doses, e.g. more than one dose, of the vaccine composition. The vaccine composition may further comprise a physiologically acceptable carrier.

In this context, "multiple doses" may mean that 1 individual administered (vaccinated) may be administered (vaccinated) more than once, or 2 individuals administered (vaccinated) may be administered (vaccinated) more than once.

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

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

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

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

The optimized amounts of components for certain vaccines can be confirmed by standard studies involving observation of appropriate immune responses in subjects. For example, the vaccination dose for humans can be determined by extrapolating the results of animal experiments. Furthermore, one skilled in the art can empirically determine the dosage as desired in the field.

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

The vaccine compositions of the present invention are useful for protecting individuals who may be infected with pneumococci and preventing pneumococcal disease by administering a pharmaceutically effective amount via systemic or mucosal route. The term "preventing" according to the invention means that all actions of infection caused by pneumococci are inhibited or delayed by administration of the vaccine composition according to the invention. By "pharmaceutically effective amount" as defined herein is meant the amount of antibody required to be produced at a level that significantly reduces the likelihood of infection by pneumococci or the severity of infection. The term "administering" in the present invention means introducing the prescribed substance into the individual by some suitable method. The vaccine composition of the present invention may be administered through the oral, nasal, rectal, transdermal or via aerosol inhalation route, but is not limited thereto. The administration may be selected from injection by intramuscular, intraperitoneal, intradermal or subcutaneous route, or mucosal administration through the oral/digestive tract, airway or genitourinary tract. As one embodiment, intranasal administration may be used to treat pneumonia or inflammation in the middle ear, and in this case, by more effectively preventing nasopharyngeal pneumococcal carrier, the inflammation may be attenuated at an early stage.

The "individual" of the vaccine composition or the pharmaceutical composition of the present invention to be administered may mean a living organism in which pathogenic bacteria can be infected or cells, tissues or culture media thereof isolated therefrom, and the organism may be a higher vertebrate, more specifically, a mammal such as a human or the like, but is not particularly limited thereto.

In other embodiments of the present invention, the composition of the present invention may be administered by a single inoculation, or two, three, four or more times at suitable intervals, but is not limited thereto. For example, the periodic vaccination schedule for infants and neonates for invasive diseases caused by streptococcus pneumoniae (Streptococcus pneumoniae) may be 2, 4, 6 and 12 to 15 months old.

In addition, the composition may further comprise one or more proteins derived from streptococcus pneumoniae (Streptococcus pneumoniae). As examples of proteins suitable for inclusion in Streptococcus pneumoniae (Streptococcus pneumoniae), not only the proteins disclosed in WO-2002/053761, but also the proteins identified in WO-2002/083855 may be included within the scope of the present invention.

In one specific example of the invention, a multivalent e.g. 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 pneumococcal vaccine composition is described comprising 2.2 μg of each polysaccharide in a total of 0.5mL vaccine composition, but the polysaccharide derived from 6B is 4.4 μg, about 29.3 μg CRM197 carrier protein, 0.5mg elemental aluminum (2 mg aluminum phosphate) adjuvant, about 4.25mg (in case no preservative is included) or about 3.5mg (in case no preservative is included) sodium chloride, about 295 μg succinate buffer, and about 3mg 2-phenoxyethanol and about 60 μg formaldehyde.

In another specific example of the invention, the serotype specific IgG concentration ratio in the serum of the rabbit in which the vaccine composition is vaccinatedThe level was higher (table 1). In addition, it was confirmed that it exhibited a specific ratioEven more excellent effect, even in functional immunogenicity confirmation test (opsonophagocytic assay) performed on it (table 2). Thus, it can be seen that the vaccine combinations of the present inventionThe composition has excellent effect on preventing pneumococcal diseases.

A further aspect of the invention is an immunogenic composition for pneumococci comprising 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 or 14 to 15 capsular polysaccharide-carrier protein conjugates. The conjugates and pneumococci are the same as described above.

The composition of the present invention comprising 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 or 14 to 15 capsular polysaccharide-carrier protein conjugates comprises capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) having 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 respectively different serotypes and when administered in vivo recognizes it as an antigen, eliciting an immune response so as to produce antibodies thereto, and thus it can be used as an immunogenic composition against pneumococci.

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

Other aspects of the invention provide for the use of a composition comprising 13 capsular polysaccharide-carrier protein conjugates in the manufacture of a vaccine composition for preventing pneumococcal disease, wherein the 13 conjugates are conjugates in which each of 13 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F is covalently coupled to the carrier protein, and the carrier protein is a CRM197 protein, and the conjugates have a structure in which the capsular polysaccharide and carrier protein are linked by an-O-C (NH) -NH-group using a cyanation method.

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

A further aspect of the invention is a method for preparing the immunogenic composition comprising the step of coupling each of 13 isolated capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F to carrier protein CRM197 such that the capsular polysaccharides and carrier proteins have a structure linked by an-O-C (NH) -NH-group.

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

[ advantageous effects ]

The multivalent pneumococcal vaccine composition of the invention comprises a multivalent conjugate of capsular polysaccharide-protein having a unique conjugate structure, and contains an optimized combination and amount of 2-phenoxyethanol and formaldehyde as preservatives therein, thereby maintaining excellent immunogenicity and having excellent stability and/or preservative ability. Thus, the vaccine compositions and immunogenic compositions of the invention may be safer and useful for preventing diseases caused by pneumococci in infants, children and adults.

Mode for the invention

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

Preparation example 1: preparation of capsular polysaccharide

1-1 preparation of cell banks

Streptococcus pneumoniae (Streptococcus pneumoniae) with 16 respectively different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B) was obtained from the commissioned organization US CDC (american disease control and prevention center) and cell banks were prepared by the following method.

Streptococcus pneumoniae (Streptococcus pneumoniae) strain was plated on blood agar medium to confirm pneumococci and to remove the medium components present. After selecting a well-grown single colony of 10 or more single colonies, it was inoculated in a liquid medium (soybean peptone (Kerry Bio-Science) or yeast extract (Bio-spring) derived medium) containing no animal-derived components therein, cultured and propagated, and then synthetic glycerol was added, thereby preparing a Research Cell Bank (RCB) containing the synthetic glycerol.

After 1 bottle of the study cell bank in which expression of polysaccharide having intrinsic serotype has been confirmed was taken out and the cells were proliferated in a liquid medium containing no animal-derived component, synthetic glycerol was added to thereby prepare a master cell bank, and after 1 bottle of the master cell bank was taken out and the cells were proliferated in a liquid medium containing no animal-derived component, synthetic glycerol was added to thereby prepare a cell bank for preparation.

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

1-2 fermentation and polysaccharide separation

1 bottle of the cell bank for preparation was thawed and inoculated in a liquid medium containing no animal-derived components. The seed culture was performed at 37±2 ℃ without agitation until a constant cell concentration (optical density, OD 600) was reached. After confirming whether the culture solution that completed the seed culture was contaminated or not, it was inoculated into a fermenter (od600=2.5±0.2) containing a liquid medium (soybean peptone (Kerry Bio-Science) or a yeast extract (Bio-spring) source medium) that did not contain an animal source component.

Then, the pH of the medium was maintained at 7.2.+ -. 0.2 with minimum agitation at 37.+ -. 2 ℃ using a sterilized potassium hydroxide solution, and main culture was performed. The concentration of cells in the culture solution and the concentration of glucose contained in the culture medium were measured by sampling from 2 hours after the start of the culture. When the glucose in the medium is depleted, the culture is complete.

After the completion of the culture, a suitable amount of sterilized 12% (w/v) sodium deoxycholate was prepared to a final concentration of 0.12% (w/v), and then it was added to the culture, thereby lysing the cells and releasing the polysaccharide bound to the cells.

1-3 purification of capsular polysaccharide

After phosphoric acid was added for a long period of time and the pH was titrated to 3.5.+ -. 0.3 to the sodium deoxycholate treated sample obtained in preparation examples 1-3, which was then allowed to react by standing for 15 hours.+ -. 3 hours, the supernatant (17,000XG, room temperature, 1 hour) was collected by centrifugation. The collected supernatant was passed through a depth filter (0.55-9.0 um) and concentrated, and buffer replacement with phosphate buffer was performed.

After the buffer replacement, the sample was passed through an activated carbon filter, and then the removal of impurities was performed by two methods:

1) Since ionic bonding to CTAB (cetyltrimethylammonium bromide) is available for 14 serotypes of serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, 19F and 23F total, the CTAB process was performed, specifically with a 10 wt% CTAB solution in an amount of 1.5 wt% (in the case of other than 23F) or 2.5% (in the case of 23F) based on the weight of the process solution to react it for 1 hour ± 10 minutes. The precipitate was then collected by centrifugation at 17,000xg for 1 hour. Subsequently, for treatment with sodium chloride (NaCl) and sodium iodide (NaI), the precipitate collected by CTAB treatment was resuspended using 200 to 300mM sodium chloride (NaCl) solution, and CTAB ions were removed using 50 wt% sodium iodide (NaI) corresponding to the amount of CTAB used. However, for serotype 3, a 350mM NaCl solution was used. After treatment with sodium chloride and sodium iodide, the supernatant was collected by centrifugation and used in the subsequent process. 2) For both serotypes 7F and 14, which were not reactive with CTAB, an aluminum phosphate gel (Algel) solution was added and reacted, followed by treatment with 10 wt.% aluminum phosphate gel solution based on total process solution and reaction for 1 hour. The supernatant obtained from centrifugation (1 hour at 17,000XG) was collected and used in the subsequent process.

Samples after completion of both types of impurity removal methods were passed through a depth filter and ultrafiltration (UF/DF) process, and then made into powder form by controlling the amounts of ethanol and sodium chloride and stored.

Preparation example 2: streptococcus pneumoniae (Strepto)coccus pneumoniae) preparation of capsular polysaccharide-protein conjugates

2-1: comparison of coupling methods of reductive amination and cyanation

The coupling procedure of reductive amination and cyanation was attempted using purified polysaccharide of serotype 9V and CRM197 carrier protein (GenBank accession number 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 tkvnsklslf feiks) to compare the coupling yields of the two coupling procedures. Specific coupling methods are described below.

2-1-1 reductive amination

11.7mg of sodium periodate was added to an undiluted solution of the polysaccharide of serotype 9V and stirred at 21 to 25 ℃ to activate the polysaccharide. After the oxidized polysaccharide was concentrated and diafiltered using a 100KDa ultrafiltration filter and WFI (water for injection), the remaining residual polysaccharide was mixed with CRM197 protein at a ratio of sugar/CRM 197 = 0.5 (weight ratio) and freeze dried. The freeze-dried complex is thawed and stabilized (equilibrated) at 21 to 25 ℃. After the complex is equilibrated by adding the complex to sodium phosphate (Na ₃ PO ₄ ) After isothermal treatment (37.+ -. 2 ℃) of 0.1M/20g of sugar in buffer to cleave it, cyanoborohydride (100 mg/mL) was added, thereby initiating the coupling reaction between the protein and sugar. After isothermal treatment at 37.+ -. 2 ℃ for about 44 to 52 hours, the temperature was lowered to 23.+ -. 2 ℃ and 1mL of 0.9% (w/v) NaCl solution was added to the reactor. Sodium borohydride solution (100 mg/mL) was added so that the molar equivalent of sodium borohydride was 1.8 to 2.2 per 1 mole of sugar, and the resulting reaction mixture was isothermally treated with stirring, consisting ofThis reduces any unreacted aldehyde present in the sugar. To the resulting glycoprotein-coupled mixture was added 5ml of 0.9% (w/v) sodium chloride solution to dilute it, and the diluted coupled mixture was dialyzed and filtered using a 100kDa MWCO membrane.

2-1-2 cyanation

Sodium chloride powder was added to an undiluted solution of serotype 9V polysaccharide prepared without a hydrolysis treatment process to prepare a 2M NaCl polysaccharide solution. To activate the polysaccharide, after adding a CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) solution to a serotype 9V polysaccharide solution so that the concentration was 0.5% (w/w) based on the polysaccharide, a polysaccharide activation reaction was induced by stirring for 15 minutes. After adding sodium hydroxide solution to the resulting reacted mixture and raising the pH to 9.5±0.1, it was stirred for 3 minutes so that the hydroxyl groups of the polysaccharide were sufficiently activated by CDAP. CRM197 was added to the polysaccharide by the polysaccharide activation process such that the ratio of CRM197 to polysaccharide was 1.0% (w/w) (CRM 197 weight/polysaccharide weight), whereby the coupling reaction was carried out for 1 hour at room temperature.

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

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

2-2. Purification of conjugates and conjugates

The preparation of conjugates of capsular polysaccharides of streptococcus pneumoniae (Streptococcus pneumoniae) and CRM197 was performed by the following process steps.

Step 1. Dissolution and hydrolysis of capsular polysaccharide

The capsular polysaccharide powder from each serotype was separately dissolved in water for injection such that the final concentration range was within the range mentioned below and filtered through a 0.45 μm filter:

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

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

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

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

The process of isothermal treatment of the solution is carried out in the pH and temperature ranges mentioned below by serotypes:

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

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

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

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

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

Step 2 coupling reaction procedure of capsular polysaccharide and CRM197

Sodium chloride powder was added to all serotypes, thereby preparing a 2M NaCl polysaccharide solution. CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) suitable for each serotype was dissolved in a ratio of 100mg CDAP per 1ml of 1/1 acetonitrile/water for injection (v/v) solution, which was added in the amounts mentioned below for the serotypes. In particular, in the present embodiment,

it was dissolved in the following ratio

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

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

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

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

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

and added to each polysaccharide solution.

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

Step 3. Termination of the coupling reaction

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

Step 4, ultra-filtration

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

Step 5, sterilizing and filtering

The solution remaining after diafiltration was diluted with buffer solution (5 mM succinate buffer solution (pH 5.8) containing 150mM NaCl) to a concentration of less than 0.4g/L in terms of polysaccharide content, and then it was filtered through a 0.22 μm filter. The filtered product was controlled during the preparation (sugar content, residual DMAP). The filtered residual solution is controlled during the preparation process, thereby determining whether additional concentration, diafiltration and/or dilution is required.

Step 6, adsorption

To the degerming filtered solution was added an aluminum salt (aluminum phosphate) so that the final concentration was 1mg/mL in terms of aluminum ion and adsorbed, and an additional salt was added to maintain the pH range of 5.5 to 6.5. The undiluted solution that completed adsorption was subjected to quality inspection, thereby confirming quality suitability, and it was refrigerated at 2 to 8 ℃ until use.

EXAMPLE 1 preparation of multivalent pneumococcal conjugate vaccine

From each of the capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B prepared in preparation 2 and CRM197 protein-coupled capsular polysaccharide-protein conjugates, the following combinations of multivalent pneumococcal polysaccharide-protein conjugates were prepared:

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

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

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

The required amount of conjugate bulk solution was calculated on the basis of the batch volume and polysaccharide concentration of the undiluted solution of conjugate. Following the addition of aluminum phosphate to the bulk solutions of the conjugates for each serotype, 0.85% (w/w) saline solution and 5mM succinate buffer solution (pH 5.8) were added, as in step 6 of preparation 2, thereby preparing a bulk solution of each conjugate containing 0.85% sodium chloride, 5mM succinate buffer solution (pH 5.8) and 1mg/mL aluminum element (concentration of aluminum element in aluminum phosphate). Finally, as preservatives, thiomersal (85 or 100 ug/ml) or 2-phenoxyethanol (2-PE) (0, 5.0 or 10.0 mg/ml) and formaldehyde (0, 10, 25, 40, 50, 80, 100, 120 or 170 ug/ml) were added and slowly mixed as the above-mentioned serotype combinations, thereby preparing a formulated multivalent pneumococcal conjugate vaccine composition. At this point, the concentration of capsular polysaccharide for each serotype of the multivalent vaccine composition was 4.4 μg/mL (8.8 μg/mL for serotype 6B only). The pH was checked and, if necessary, adjusted to pH 5.8. The prepared undiluted solution of the formulated final vaccine composition was filled into borosilicate glass bottles type 1. The bottled vaccine composition is stored at 2 to 8 ℃.

Example 2: estimation of immunogenicity of multivalent pneumococcal conjugate vaccine

A multivalent pneumococcal vaccine composition (designated LBVE 013) containing 2.2 μg of each capsular polysaccharide (4.4 μg of serotype 6B capsular polysaccharide only), about 29.3 μg of CRM197 carrier protein, 0.5mg of elemental aluminum (2 mg aluminum phosphate) adjuvant, about 4.25mg sodium chloride, about 295 μg succinate buffer, about 3mg 2-phenoxyethanol and about 60 μg formaldehyde in a total of 0.5mL vaccine formulation was selected as a 13 valent (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) vaccine in the vaccine composition prepared in example 1, thereby confirming its ability to induce an immune response in rabbits. This immunogenicity was demonstrated by antigen-specific ELISA at serotype IgG concentrations and by opsonophagocytosis assay (OPA) at antibody function.

Vaccine composition LBVE013 or as positive control groupAt weeks 0, 2 and 4, the muscles of New Zealand white rabbits were immunized with the planned clinical dose of human beings (2.2. Mu.g of each polysaccharide, with the exception of 6B 4.4. Mu.g), and serum was collected at intervals of 2 weeks after inoculation. IgG measurements obtained for the collected serum using ELISA are shown in table 1. The detailed description is as follows.

2-1 serotype specific IgG concentration measurement

A total of 13 capsular polysaccharides for each serotype were treated at 5 μg/well on 96-well plates, whereby it was coated at room temperature for 16 hours. To minimize the serum nonspecific antigen-antibody reaction, serum from each individual was collected in the same amount, thereby pooling the same group of serum. After mixing the serum pool with 333.3. Mu.g/mL of C-PS (cell wall polysaccharide; SSI (status serum)m Institute)) and 333.3 μg/mL of capsular polysaccharide of serotype 22F (PnPs 22F) at room temperature for 30 minutes to adsorb it, it was diluted with a buffer solution for antibody dilution comprising tween 20 at a suitable dilution ratio (about 1:100-1:40000). The coated plate was washed 4 times with a buffer solution for washing, and 50. Mu.l of the adsorbed and diluted serum was placed in a coated well plate, which was then reacted at room temperature for 1 hour. The wells of the reacted wells were washed 4 times by the same method, and goat anti-rabbit IgG antibody-HRP conjugate (1:20000) was placed in each well, and then it was reacted at room temperature for 30 minutes. Plates were washed 4 times by the same method and 100. Mu.l of stabilized TMB substrate solution (3, 3', 5' -tetramethylbenzidine; sigma, st.Louis, MO, USA) was placed in each well at room temperature, and then it was reacted at room temperature for 15 minutes. After stopping the reaction by placing 100. Mu.l of a 1N sulfuric acid solution, absorbance at 450nm was measured using 650nm as a reference wavelength. For objective evaluation of immunogenicity, the same method was used as a control group for analysis A collected blood sample.

The results are disclosed in table 1 below.

TABLE 1

As shown in table 1, it was confirmed that the immunogenic response pattern according to serotypes of the multivalent (13-valent) pneumococcal vaccine composition (LBVE 013) was definitely different fromIs a mode of (2). And->In contrast, higher threshold levels were exhibited in all serotypes, particularly serotypes 1, 6B, 7F, 9V, 14 and 19F, in rabbits vaccinated with the 13-valent vaccine composition of this example (LBVE 013)Ratio->The effect is 2-6 times higher.

2-2. Functional immunogenicity confirmation assay (opsonophagocytosis assay, OPA)

The function of the serotype-induced antibodies was assessed by OPA analysis of serum obtained from rabbits.

Specifically, the same amount of serum was collected from each individual, thereby pooling the same group of serum. Streptococcus pneumoniae (Streptococcus pneumoniae) of each serotype was cultivated in THY medium (Todd-Hewitt broth containing 2w% yeast extract) and diluted to 200 to 300CFU/10 μl using conditioning buffer. Mu.l of diluted serum and 10. Mu.l of diluted Streptococcus pneumoniae (Streptococcus pneumoniae) were mixed and reacted at room temperature for 30 minutes. Then, 50 μl of the mixture of differentiated HL-60 cells and complement (cells: complement=4:1) was added and mixed in CO ₂ The reaction was carried out in the medium (37 ℃) for 45 minutes.

Cell phagocytosis was stopped by lowering the temperature and 10. Mu.l of the reaction solution was plated in agar medium which had been dried for 30 to 60 minutes. Then, it is put in CO ₂ The colonies were cultured in the medium (37 ℃) for 12 to 18 hours and counted. OPA threshold is expressed as dilution at which 50% death is observed.

The results obtained are shown in table 2 below:

TABLE 2

In table 2, for example, OPA titer is expressed as 2187 which is a case where the 50% level is not reached even in the most dilute portion compared to the negative control group, and it means that the threshold value is remarkably high. Through the above test results, it is confirmed thatThe multivalent pneumococcal vaccine composition of this example is implementedHas a remarkably excellent serum IgG threshold. Thus, the multivalent pneumococcal vaccine composition of this embodiment can be very usefully used for preventing diseases caused by pneumococci.

Reference example: procedure and criteria for vaccine bactericidal ability test

The bactericidal capacity test of the vaccine was performed according to the EP-B standard, which is required for vaccine products by the World Health Organization (WHO) of the united states pharmacopeia (USO) and the European Pharmacopeia (EP) in this example.

If the contents of the test are summarized in detail, 2 bacteria Pseudomonas aeruginosa (Pseudomonas aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), candida albicans (ATCC No.10231, CA) and Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) were combined in total to form four pathogens 10 ⁵ To 10 ⁶ CFU/mL (CFU; colony forming units) were inoculated at 0 to the vaccine compositions, respectively. Colony numbers were counted at 3 to 5 days by sampling at 24 hours, 7 days, 14 days and 28 days and culturing them in solid medium.

For the results according to the above method, the criteria of the bactericidal capacity of EP-B and of the pharmacopoeias of each country are shown in Table 3 below.

TABLE 3

* NR: not recovered

* NI: is not increased

As can be seen in table 3, EP requirements are more stringent than the United States Pharmacopeia (USP) or Japanese Pharmacopeia (JP) and are classified according to formulation into class a and B, and WHO requires a level of vaccine product of EP-B (EP 5.1.3. Preservative efficacy of antimicrobial (Efficacy of antimicrobial perserbation), USP 37-51, antimicrobial effectiveness test (Antimicrobial effectiveness testing)).

In this example, to develop a multivalent pneumococcal polysaccharide-protein conjugate vaccine, the vaccine preservatives thimerosal and 2-PE commonly used in the art were added, thereby performing a bactericidal capacity test, and confirming that the criteria for bactericidal capacity were not met with thimerosal alone or low concentrations of 2-PE.

Furthermore, since other companies have filed patent applications for cases where high concentrations of 2-PE (7 mg/mL or more) are used, it cannot be used in our company.

Thus, as a result of experiments in progress, the inventors of the present invention have obtained the following results, thereby developing a novel composition that can minimize the content of 2-PE and enhance the bactericidal effect so as to meet the bactericidal capacity standard without violating the patents of other companies, as a preservative for multivalent pneumococcal protein conjugate vaccine.

Example 3: screening of bacterial killing ability of multivalent pneumococcal polysaccharide-protein conjugate vaccine

Among four bacteria of 2 bacteria Pseudomonas aeruginosa (Pseudomonas aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), candida albicans (ATCC No.10231, CA) and Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN), a known Staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA) which is not easily killed by 2-PE was selected as a bacteria for screening for bactericidal activity. The purpose of the experiment was to select a combination of 2-PE and formaldehyde that meets EP-B by estimating the bactericidal capacity using a sample taken at 24 hours. Thus, the test was carried out according to the test method of EP-B.

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

TABLE 4

As can be seen in Table 4, the bactericidal capacity of SA from the combination of 2-PE 5.0mg/mL with formaldehyde 100. Mu.g/mL passed the EP-B standard. Based on the results, a combination of about 5.0mg/mL 2-PE with more than 100. Mu.g/mL formaldehyde was demonstrated to be suitable as a preservative for multivalent pneumococcal polysaccharide-protein conjugate vaccines.

Example 4: bacterial killing test of multivalent pneumococcal polysaccharide-protein conjugate vaccine 1

The bactericidal test was carried out according to the test method of EP-B. Four bacteria of Pseudomonas aeruginosa (Pseudomonas aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), candida albicans (ATCC No.10231, CA) and Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) were added at 10 ⁵ To 10 ⁶ CFU/mL (CFU; colony forming units) was inoculated at 0 to multivalent pneumococcal protein conjugate vaccine formulations of vaccine compositions 13-15, respectively. The number of colonies was counted at 3 to 5 days by sampling at 0, 24 hours, 7 days and 28 days and culturing it in a solid medium, thereby calculating the log of colony reduction. Fungi and yeasts were sampled at 0, 14 and 28 days and cultured in a solid medium, and then the colony numbers were counted at 3 to 5 days, thereby calculating the fungiLog of the reduction. The results are shown in table 5.

TABLE 5

* NR: not recovered

* NI: is not increased

As shown in Table 5, as a result of confirming the bactericidal ability of four kinds of bacteria among 2 kinds of bacteria Pseudomonas aeruginosa (Pseudomonas aeruginosa) (ATCC No.9027, PA), staphylococcus aureus (Staphylococcus aureus) (ATCC No.6538, SA), candida albicans (ATCC No.10231, CA) and Aspergillus niger (Aspergillus niger) (ATCC No.16404, AN) in multivalent pneumococcal polysaccharide-protein conjugate vaccine compositions 13 to 15 containing therein each of the serotypes of 13, 15A and 15B, the bactericidal ability of 4 kinds of bacteria satisfies EP-B standard in all of 13, 15A and 15B regardless of the serotypes. From the results, it was demonstrated that the bactericidal capacity of EP-B standard was met with preservatives of multivalent pneumococcal polysaccharide-protein conjugate vaccine comprising about 5mg/mL 2-PE and about 100 μg/mL formaldehyde.

In the art pneumococcal protein conjugate vaccines typically use more than 7mg/mL of 2-PE as preservative, but the results together with the results of example 3 show that the bactericidal capacity of EP-B can also be achieved with a content of 2-PE as low as 5mg/mL with the addition of formaldehyde in an amount of more than 100 μg/mL. Thus, in the present invention, a multi-dose composition can be provided in which an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) is used as a preservative for multivalent pneumococcal polysaccharide-protein conjugate vaccines, and a strong and effective bactericidal capacity is maintained.

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

Based on the results of example 4, a new composition of multivalent pneumococcal polysaccharide-protein conjugate vaccine composition 16 was prepared. Specifically, the vaccine composition 16 is prepared by the following method: 14 polysaccharide-protein conjugates (serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) and 1mg/mL aluminum phosphate (in aluminum concentration) were placed and stirred for thorough mixing. 5mM succinate and 0.85% (w/v) sodium chloride were dissolved in distilled water for injection, whereby the sterilized filtered buffer was mixed. After final homogenization by adding 2-PE at a concentration of 6.0mg/mL and formaldehyde at a concentration of 120. Mu.g/mL, the final pH was adjusted to 5.8. The concentration of each serotype in the vaccine composition prepared was likewise 4.4. Mu.g/mL (only 6B was 8.8. Mu.g/mL).

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

In summary, the combination of 2-PE and formaldehyde illustrated in the examples of the present invention is demonstrated to be an excellent composition that effectively maintains the bactericidal activity of multivalent pneumococcal polysaccharide-protein conjugate vaccine for a long period of time, and it can be usefully employed to provide a multi-dose composition in which the bactericidal ability of multivalent polysaccharide-protein conjugate vaccine is improved or maintained for an extended period of time.

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

<110> LG chemistry from Kagaku Co., ltd (LG CHEM, LTD.)

<120> multivalent Streptococcus pneumoniae vaccine compositions

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<150> KR 10-2017-0032643

<151> 2017-03-15

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<170> KoPatentIn 3.0

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<223> CRM197 protein

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Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu

1 5 10 15

Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile

20 25 30

Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp

35 40 45

Asp Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala

50 55 60

Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys

85 90 95

Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr

100 105 110

Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe

115 120 125

Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly

130 135 140

Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln

165 170 175

Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val

180 185 190

Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp

195 200 205

Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His

210 215 220

Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser

225 230 235 240

Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu

245 250 255

Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro

260 265 270

Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln

275 280 285

Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala

290 295 300

Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly

305 310 315 320

Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu

325 330 335

Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val

340 345 350

Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu

355 360 365

Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly

370 375 380

His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn

385 390 395 400

Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly

405 410 415

His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly

420 425 430

Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys

435 440 445

Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala

450 455 460

Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val

465 470 475 480

Gly Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser

485 490 495

Ser Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val

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

1. A multivalent vaccine composition against pneumococci, comprising:

(i) A capsular polysaccharide-carrier protein conjugate,

(ii) 5mg/mL or more and less than 6mg/mL of 2-phenoxyethanol (2-PE), and

(iii) Formaldehyde (HCHO) of 100 μg/mL to 170 μg/mL,

wherein the capsular polysaccharide comprises 13 to 24 capsular polysaccharides derived from streptococcus pneumoniae (Streptococcus pneumoniae) serotype.

2. The multivalent vaccine composition against pneumococci according to claim 1, wherein the capsular polysaccharide comprises 13 to 24 capsular polysaccharides selected from the group consisting of: capsular polysaccharides derived from streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

3. The multivalent vaccine composition against pneumococci of claim 2, wherein the capsular polysaccharide comprises

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

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

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

15 capsular polysaccharides derived from streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2 and 15B; or (b)

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

4. The multivalent vaccine composition against pneumococcus of claim 1, wherein the carrier protein is CRM197 protein.

5. The multivalent vaccine composition against pneumococci of claim 1, wherein the capsular polysaccharide-carrier protein conjugate has a structure in which the capsular polysaccharide and carrier protein are linked by-O-C (NH) -NH-groups using a cyanation method.

6. The multivalent vaccine composition against pneumococci according to claim 5, wherein the cyanation method is performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.

7. The multivalent vaccine composition against pneumococci of claim 1, comprising:

based on 1 part by weight of capsular polysaccharide from serotype 1,

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

1.8 to 2.2 parts by weight of capsular polysaccharide from serotype 6B.

8. The multivalent vaccine composition against pneumococcus according to any one of claims 1 to 7 for multiple administration.

9. A pharmaceutical composition for preventing pneumococcal infection or pneumococcal infection disease comprising a multivalent vaccine composition against pneumococci according to any one of claims 1 to 7.

10. The pharmaceutical composition of claim 9, wherein the pneumococcal infection disease is pneumonia.

11. A method for preparing a multivalent vaccine composition against pneumococci, the method comprising:

(1) Preparation of capsular polysaccharide-protein conjugates of Streptococcus pneumoniae, and

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

wherein the capsular polysaccharide comprises 13 to 24 capsular polysaccharides derived from streptococcus pneumoniae serotypes, and

the amount of 2-phenoxyethanol used is 5mg/mL or more and less than 6mg/mL, and the amount of formaldehyde is 100 μg/mL to 170 μg/mL.

12. The method for preparing a multivalent vaccine composition against pneumococci according to claim 11, wherein step (1) of preparing a capsular polysaccharide-protein conjugate comprises the step of performing a cyanation method to link the capsular polysaccharide and protein through-O-C (NH) -NH-.