WO2018169303A1 - Composition vaccinale polyvalente contre streptococcus pneumoniae - Google Patents

Composition vaccinale polyvalente contre streptococcus pneumoniae Download PDF

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Publication number
WO2018169303A1
WO2018169303A1 PCT/KR2018/002983 KR2018002983W WO2018169303A1 WO 2018169303 A1 WO2018169303 A1 WO 2018169303A1 KR 2018002983 W KR2018002983 W KR 2018002983W WO 2018169303 A1 WO2018169303 A1 WO 2018169303A1
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Prior art keywords
vaccine composition
protein
polysaccharide
capsular
streptococcus pneumoniae
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PCT/KR2018/002983
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English (en)
Korean (ko)
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조성제
최숙영
박혜미
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주식회사 엘지화학
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Priority to CN201880025013.0A priority Critical patent/CN110520154B/zh
Publication of WO2018169303A1 publication Critical patent/WO2018169303A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine

Definitions

  • the present invention relates to a pneumococcal ( Streptococcus pneumoniae ) vaccine composition, and more specifically, (i) capsular polysaccharide-protein conjugate, (ii) 2-phenoxyethanol , 2-PE), and (iii) formaldehyde (HCHO) and a method for preparing the same.
  • Pneumococcus Streptococcus pneumonia ; Pneumococcus
  • Streptococcus pneumonia is a gram-positive and hemolytic streptococcus that is a major cause of meningitis, pneumonia, and severe invasive infectious diseases in infants, children, and the elderly worldwide. More than 1.6 million people die each year from pneumococcal disease (2008 International Health Organization), and the incidence of invasive infectious diseases caused by pneumococcal in children under 5 years old and elderly people 65 years or older with low immunity high.
  • Pneumococci are classified into more than 90 serotypes, depending on the structural and immunological properties of the capsular polysaccharides, the main pathogenic factors surrounding their outer membranes. It is known to be associated with 80-90% pathogenicity.
  • the only host of pneumococci is humans, and they usually exist in colonies in the nasopharynx of healthy normal people (20-40% in infants, 5-10% in adults).
  • the US Centers for Disease Control and Prevention (CDC) estimated that approximately 2.1 million children under 5 years of age died from pneumonia, and 1.2 million of them died from developing countries alone annually.
  • pneumococcal meningitis and sepsis are reported to be about 3,000 and 50,000 each year (Peters TR, Poehling KA et al. Invasive pneumococcaldisease. JAMA 2007; 297: 1825-6).
  • pneumoACTION which is a database of pneumococcal disease, showed that 24,047 cases of pneumococcal infections occurred in Korean children in 2000, and 47 of them died (www.pneumoadip.org).
  • pneumococci are the most common cause of invasive infections (43.7%) in infants aged 3 to 59 months. appear.
  • pneumococci causing invasive infectious diseases all over the world, multidrug resistant bacteria that are resistant to not only penicillin but also three or more drugs are increasing, further increasing the difficulty of treating pneumococcal infectious diseases.
  • multivalent pneumococcal polysaccharide vaccines have been developed and approved since 1977, and these capsular polysaccharide vaccines have proven useful in preventing pneumococcal disease in elderly and high-risk patients.
  • the immune system since the maturity of the immune system is lower than that of adults, when only the polysaccharide vaccine is received, the immune system does not recognize the polysaccharide antigen as an external invading factor, so it is difficult to expect a role as a vaccine.
  • a 7-valent pneumococcal conjugate vaccine a capsular polysaccharide-protein conjugated vaccine conjugated with a carrier protein that increases immunogenicity to polysaccharide antigens ( 7vPnC, Prevenar®) has been developed and used and many data have been reported to be effective for the prevention of invasive diseases and otitis media in infants and children.
  • 7vPnC polysaccharide antigens
  • the use of the 7-valent vaccine induced a decrease in invasive disease caused by vaccine serotypes used in the vaccine, but also showed a relative increase in pneumococcal disease caused by some non-vaccine serotypes.
  • Synflorix® a 10-valent capsular polysaccharide-protein conjugate vaccine
  • Prevenar13® a 13-valent pneumococcal conjugate vaccine, in which 6 serotypes were added to the base serotype of Prevena®.
  • has been developed and is currently commercially available, but the possibility that the efficacy as a vaccine may not be sufficient for some of the serotypes included is reported by Andrews NJ et al, (2014) Lancet Infec Dis (14) 839; EMEA Assessment Report for Prevenar 13 (2009) EMA / 798877/2009], there is a continuing need for the development of new vaccine formulations that show higher and stable potency.
  • Vaccine dose injections should use preservatives to prevent microbial contamination.
  • Mixed vaccine products exported to underdeveloped countries through the United Nations have been favored in multi-dose products, including preservatives, due to the country's environment, distribution methods and costs.
  • Preservatives used in vaccine products include chimerosal, phenoxyethanol (2-PE), phenol, and the like, and the amount of preservatives commonly used in the art.
  • chimerosal is used at a concentration of 10 ⁇ g / mL
  • 2-PE is used at a concentration of 5 mg / mL
  • the multidose vaccine product including this is EP-B (European Pharmacopoeia B category) or USP (US Pharmacopoeia).
  • the product must be passed the standard antiseptic test.
  • Chimerosal (Thimerosal, Thiomersal, merthiolate) is an ethyl mercury derivative compound that has been used as a preservative for multidose vaccine injections since the early 1930s. Chimerosal has been used for the purpose of preventing the growth of contaminating microorganisms and maintaining aseptic status when storing or using vaccine products, and a number of pentavalent liquid mixed vaccines (D, T, P, Hib) that have obtained WHO PQ (Prequalified). , HBsAg) contains chimerosal as a preservative.
  • 2-Phenoxyethanol (2-PE) is mainly used as a preservative in cosmetics and transdermal drugs, and also as a preservative in vaccine injections.
  • Examples include (i) capsular polysaccharide-protein conjugates of Streptococcus pneumoniae , (ii) 2-phenoxyethanol (2-PE), and (iii) formaldehyde It provides a pneumococcal vaccine composition comprising (Formaldehyde, HCHO).
  • the vaccine composition may be a multiple dose vaccine composition for multiple administrations.
  • Another example provides a pharmaceutical composition for the prevention or treatment of pneumococcal infection diseases comprising the pneumococcal vaccine composition described above.
  • the prophylactic or therapeutic pharmaceutical composition may be a multiple dose pharmaceutical composition for multiple administrations.
  • kits for preparing a capsular polysaccharide-protein conjugate of Streptococcus pneumoniae and the capsular polysaccharide-protein conjugate with 2-phenoxyethanol (2-PE).
  • a method for preparing stability and / or preservative (or antiseptic) enhanced pneumococcal vaccine composition or the stability and / or preservative (or preservative) of pneumococcal vaccine comprising mixing formaldehyde (HCHO).
  • HCHO formaldehyde
  • the vaccine composition may be a multiple dose vaccine composition for multiple administrations.
  • Preparing the capsular polysaccharide-protein conjugate may include linking the capsular polysaccharide and the carrier protein through -OC (NH) -NH- by performing a cyanylation method.
  • Examples include (i) the capsular polysaccharide-protein conjugate of Streptococcus pneumoniae , (ii) 2-Phenoxyethanol (2-PE), and (iii) Formaldehyde (HCHO). It provides a pneumococcal vaccine composition or pneumococcal immunogenic composition comprising.
  • the pneumococcal immunogenic composition refers to a composition that induces an immune response against pneumococcal, and unless otherwise stated, is used in the same sense as the pneumococcal vaccine composition.
  • composition comprising (predetermined components)” may mean comprising essentially or containing components other than the components described. .
  • the pneumococcal vaccine composition provided herein includes a capsular polysaccharide of Streptococcus pneumoniae and 2-phenoxyethanol and formaldehyde as preservatives, thereby inducing an immune response against pneumococci. It may be an immunogenic composition with enhanced stability and / or preservation (preservation) that (immunogenicity) is maintained for a long time.
  • the capsular polysaccharide may be a capsular polysaccharide derived from Streptococcus pneumoniae .
  • the capsular polysaccharide is derived from two or more of the Streptococcus pneumoniae serotypes, for example, five or more, seven or more, nine or more, eleven or more, 13 or more, or 14 or more serotypes. It may be a capsular polysaccharide.
  • the capsular polysaccharide is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 of the Streptococcus pneumoniae serotypes. It may be one containing a capsular polysaccharide derived from 19, 14 to 17, or 14 to 15 serotypes.
  • the capsular polysaccharide is Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A.
  • 13 to 24 species 13 to 19 species, 13 to 17 species, 13 to 15 species, 14 to 24 species, 14 to 19 species selected from the group consisting of 19F, 20, 22F, 23F, and 33F. It may include a capsular polysaccharide derived from a species, 14 to 17 species, or 14 to 15 serotypes.
  • the capsular polysaccharide is 13 capsulomas derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.
  • the capsular polysaccharide-protein conjugate may be one in which the capsular polysaccharides derived from two or more serotypes described above are individually conjugated with proteins, respectively, through conventional methods, such as covalent bonds.
  • the conjugate has a structure in which each capsular polysaccharide (specifically, the hydroxy group of the polysaccharide) and the protein (specifically, the amino group of the protein) are linked via -OC (NH) -NH- using a cyanylation method. ([Polysaccharide] -OC (NH) -NH- [protein]).
  • the degree of structural modification of the capsular polysaccharide is less likely to maintain immunogenicity even after conjugation. It may be more advantageous.
  • certain serotypes eg 19F
  • the hexasaccharide ring structure may be cleaved and open, resulting in a decrease in immunogenicity.
  • this problem does not occur when the protein is conjugated with cyanylation.
  • the protein can be a carrier protein, for example, a protein that is nontoxic and nonreactive and can be obtained in sufficient amount and purity. Threatening polysaccharides from various serotypes can each be conjugated with the same or different proteins, such as the same protein.
  • the protein may be CRM197 protein.
  • CRM197 protein is a non-toxic variant of diphtheria toxin isolated from cultures of Corynebacterium diphtheria strain C7 (CRM197; such as grown in casamino acid and yeast extract base medium), ie, toxoid.
  • CRM197 protein is purified from Corynebacterium diphtheria strain C7 culture via ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography, or described in conventional methods such as US Pat. No. 5,614,382 (incorporated herein by reference). It may be obtained recombinantly with reference to the method.
  • the CRM197 protein is GenBank Accession No. It may comprise an amino acid sequence of 1007216A or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology with the sequence.
  • Denatured toxins derived from diphtheria other than Corynebacterium diphtheria strain C7 can also be used as carrier proteins.
  • Inactivated bacterial toxins such as tetanus toxin modified, modified pertussis toxin, cholera toxin-modified (e. G., International Patent Application Publication No. AS as described in WO2004 / 083251 No.), Escherichia coli (E. coli), LT, Escherichia Chitocoli ST, and exotoxin A from Pseudomonas aeruginosa;
  • Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding protein, pneumolysine, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), group A or C5a peptidase from Group B streptococci, Haemophilus influenzae protein D;
  • OMPC outer membrane complex c
  • porins porins
  • transferrin binding protein pneumolysine
  • pneumococcal surface protein A PspA
  • pneumococcal adhesin protein PsaA
  • group A or C5a peptidase from Group B streptococci Haemophilus influenzae protein D
  • Purified protein derivatives of ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), tuberculin;
  • Variants of diphtheria toxins such as CRM197, CRM173, CRM228, CRM45 (modified toxins);
  • It may be one or more selected from the group consisting of.
  • the capsular polysaccharide-protein conjugate is derived from two or more of the Streptococcus pneumoniae serotypes, eg, at least 5, at least 7, at least 9, at least 11, at least 13, or at least 14 serotypes. It may be a polyvalent polysaccharide-protein conjugate comprising a conjugate to which each capsular polysaccharide and a carrier protein described above, such as CRM197 protein, are conjugated. In one embodiment, the capsular polysaccharide-protein conjugate is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 of the Streptococcus pneumoniae serotypes.
  • the capsular polysaccharide-protein conjugate is a Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 selected from the group consisting of 18C, 19A, 19F, 20, 22F, 23F, and 33F.
  • 13- to 24-valent, 13-valent comprising conjugates conjugated to each of the capsular polysaccharides derived from the species-19 species, 14-17 species, or 14-15 species serotypes and a carrier protein such as CRM197 protein.
  • a carrier protein such as CRM197 protein.
  • the capsular polysaccharide-protein conjugate is 13 derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.
  • 13-valent polysaccharide-protein conjugates comprising 13 conjugates of each species of capsular polysaccharide and a transport protein such as CRM197 protein; 14-valent polysaccharide-protein conjugates derived from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; Each of 15 capsular polysaccharides and carrier proteins from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F, For example, a 15-valent polysaccharide-protein conjugate comprising 15 conjugates to which CRM197 protein is conjugated; Each of 15 capsular polysaccharides and carrier proteins from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B, For example, a 15
  • the pneumococcal vaccine composition provided herein is a Pneumococcal conjugate vaccine (PCV) comprising a capsular polysaccharide-protein conjugate.
  • PCV Pneumococcal conjugate vaccine
  • the pneumococcal vaccine composition is each derived from two or more of the Streptococcus pneumoniae serotypes, eg, at least 5, at least 7, at least 9, at least 11, at least 13, or at least 14 serotypes. It may be a multivalent pneumococcal vaccine composition comprising a polysaccharide-protein conjugate conjugated with a capsular polysaccharide of and a carrier protein described above, such as CRM197 protein.
  • the pneumococcal vaccine composition is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, among Streptococcus pneumoniae serotypes, 13 to 24, 13 to 19 species in which each capsular polysaccharide derived from 14 to 19, 14 to 17, or 14 to 15 serotypes and a carrier protein such as CRM197 protein are conjugated.
  • 13 to 17 species, 13 to 15 species, 14 to 24 species, 14 to 19 species, 14 to 17 species, or 14 to 15 species comprising the polysaccharide-protein conjugate , 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition.
  • the pneumococcal vaccine composition is Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C. , 13A to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 selected from the group consisting of 19A, 19F, 20, 22F, 23F, and 33F.
  • 13 to 24 valent comprising a polysaccharide-protein conjugate conjugated with a capsular polysaccharide and a carrier protein such as CRM197 protein, each derived from to 19, 14 to 17, or 14 to 15 serotypes, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition.
  • a carrier protein such as CRM197 protein
  • the pneumococcal vaccine composition is
  • each of 13 capsular polysaccharides derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F and a transport protein, such as A trivalent pneumococcal vaccine composition comprising 13 polysaccharide-protein conjugates to which CRM197 protein is conjugated;
  • a 14-valent pneumococcal vaccine composition comprising 14 polysaccharide-protein conjugates to which CRM197 protein is conjugated;
  • a 15-valent pneumococcal vaccine composition comprising 15 polysaccharide-protein conjugates to which carrier proteins such as CRM197 protein are conjugated;
  • a 15-valent pneumococcal vaccine composition comprising 15 polysaccharide-protein conjugates to which carrier proteins such as CRM197 protein are conjugated; or
  • 17 different capillaries derived from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F 17-valent pneumococcal vaccine composition comprising 17 polysaccharide-protein conjugates each conjugated to a polysaccharide and a carrier protein such as CRM197 protein
  • Multivalent pneumococcal vaccine compositions provided herein such as 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 14 19-, 14-17 or 14- to 15-valent pneumococcal vaccines have significantly superior titers compared to previously developed multivalent vaccines (such as 13-valent vaccines), and can prevent and / or prevent pneumococcal infectious diseases. A very good effect can be expected for treatment.
  • the pneumococcal vaccine composition may be a formulation formulated in a single dose for multiple administrations or in multiple doses (multi-dose) for multiple administrations.
  • a "multidose” is a vaccine dose capable of more than one (eg, two or more) administrations (inoculation) to one administration (inoculation) individual, or one or one of two or more administrations (inoculation) individuals.
  • formulation unit can be meant comprising a vaccine dose that can be administered (inoculated) more than (eg, two or more times).
  • the pneumococcal vaccine composition includes a preservative in addition to two or more serotypes of pneumococcal capsular polysaccharide-protein conjugates.
  • Preservatives usable in such vaccine compositions include 2-phenoxyethanol, formaldehyde, chlorobutanol, m-cresol, methylparaben, propylparaben, benzetonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol It may be one or more selected from the group consisting of phenol, chimerosal, mercury nitrate, and the like.
  • the preservative is a multivalent pneumococcal vaccine composition as described above, for example, 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, As a form optimized for the 14-19, 14-17, or 14--15 pneumococcal vaccine composition, it may include 2-phenoxyethanol and formaldehyde. As such, by containing 2-phenoxyethanol as formaldehyde together with formaldehyde, the improved preservative effect is achieved by synergistic action of the two components while reducing the amount of 2-phenoxyethanol to reduce toxicity and / or side effects. can do.
  • the content of 2-phenoxyethanol contained in the multivalent pneumococcal vaccine composition of the present invention is less than 10 mg / ml, less than 7 mg / ml, 6 mg / ml or less, or 5 mg / ml or less, such as 4 mg / ml or more and less than 10 mg / ml, 4 mg / ml to 7 mg / ml, 4 mg / ml or more but less than 7 mg / ml, 4 to 6 mg / ml, 4.5 mg / ml to 6 mg / ml, 5 mg / ml to 6 mg / ml, 4 to 5 mg / ml, or 4.5 mg / It may be ml to 5 mg / ml (as used herein, “to” as used in the numerical range expression indicates a numerical range below the lower limit or above the upper limit.
  • the content of 2-phenoxyethanol in the vaccine composition is preferably above the lower limit of the above range in order to exhibit an effective antiseptic effect, and below the upper limit or below the upper limit of the above range in order to have no toxicity and / or side effects or to be acceptable. .
  • the content of formaldehyde contained in the multivalent pneumococcal vaccine composition of the present invention is 90 to 200 ⁇ g / mL, 90 to 190 ⁇ g / mL, 90 to 180 ⁇ g / mL, 90 to 170 ⁇ g / mL, 100 to 200 ⁇ g / mL , 100 to 190 ⁇ g / mL, 100 to 180 ⁇ g / mL, or 100 to 170 ⁇ g / mL.
  • the content of formaldehyde in the vaccine composition is preferably higher than the lower limit of the above range in order to exhibit an effective antiseptic effect, and below the upper limit of the above range in order to have no toxicity and / or side effects or be acceptable.
  • the vaccine composition may be at least about 3 months, at least about 6 months, at least about 1 year, at least about 1.5 years at normal storage conditions, such as at temperatures ranging from 2 ° C. to 8 ° C., 20 ° C. to 25 ° C., or about 37 ° C. , For about 2 years or more, or for about 2.5 years or more.
  • the stability of the vaccine composition herein means that the vaccine composition maintains its original antigenicity (immunogenicity) at an equivalent level, and / or that each component is maintained without degradation or loss, and / or infections such as bacteria / viruses, etc. May mean none.
  • Another example provides a pharmaceutical composition for preventing or treating pneumococcal infection or pneumococcal infection disease, including the multivalent pneumococcal vaccine composition described above.
  • Another example is the prevention of pneumococcal infection or pneumococcal infection disease comprising administering a pharmaceutically effective amount of the multivalent pneumococcal vaccine composition described above to a subject in need of prevention or treatment of pneumococcal infection or pneumococcal infection disease or Provide a method of treatment.
  • the pneumococcal vaccine composition or pneumococcal vaccine composition used in the prophylactic or therapeutic method included in the pharmaceutical composition may be a single dose pharmaceutical composition for single administration or a multi-dose pharmaceutical composition for multiple administration.
  • the pneumococcal disease refers to all diseases caused by pneumococcal infection, and may be pneumonia, otitis media, sinusitis, bacteremia and the like.
  • "Pneumonia” is a type of acute inflammatory disease of the lung parenchyma.
  • the main infectious agents are Streptococcus pneumoniae and Klebsiella. pneumoniae ).
  • pneumococcal pneumonia accounts for about 50% of all pneumonia, severe chills, fever, cough and chest pain, sputum is often bloody, complications that can cause pleurisy, meningitis, endocarditis, peritonitis ( Diagn. Microbiol.Infect.Dis., 2001, 39: 181-185).
  • pneumococcus in the present invention refers to Streptococcus pneumoniae and is generally a commensal organism that colonizes the mucosal surface of human nasopharynx. If the host's factor allows access to the lower respiratory tract of the organism, then a vigorous inflammatory response follows, which causes dense consolidation when the alveolar space fills the exudate, resulting in pneumonia May cause.
  • the pneumococci can synthesize more than 90 structurally unique capsular polysaccharides, and the serotypes of pneumococci are classified according to the structural and immunological characteristics of these capsular polysaccharides.
  • the immune response may be different depending on the type of capsular polysaccharide, that is, the serotype of pneumococcal from which capsular polysaccharide is derived.
  • a vaccine composition for preventing pneumococcal can be prepared using the capsular polysaccharide.
  • the term "antigen” refers to a substance capable of specifically inducing an immune response when the substance is invaded into the body.
  • Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C 13 to 24 capsular polysaccharides derived from, 19A, 19F, 20, 22F, 23F, and 33F may act as antigens, respectively.
  • Another example provides a method of making a pneumococcal vaccine composition having enhanced stability and / or preservative (or antiseptic) or a method of enhancing the stability and / or preservative (or antiseptic) of a pneumococcal vaccine.
  • the method is
  • the capsular polysaccharide, the protein, the conjugate, and the content of 2-phenoxyethanol and formaldehyde are as described above.
  • the capsular polysaccharide can be prepared by standard techniques known to those skilled in the art, and are not particularly limited thereto.
  • the capsular polysaccharide can be reduced in size through hydrolysis to reduce viscosity and induce effective immunogenicity.
  • (1) preparing the capsular polysaccharide-protein conjugate may include linking the capsular polysaccharide and the protein through -O-C (NH) -NH- by performing a cyanylation method.
  • the (1) preparing the capsular polysaccharide-protein conjugate
  • the preparing of the capsular polysaccharide-protein conjugate (1) may be performed by linking the capsular polysaccharide and the protein through -OC (NH) -NH- by performing the (iii) cyanylation method. Thereafter, one or more steps selected from the ultrafiltration step, the sterile filtration step, and the adsorption step can be further performed.
  • the cyanylation method may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.
  • Streptococcus pneumoniae having 13 or 15 different serotypes was lysed using sodium deoxycholate, respectively, to release polysaccharides bound to cells. Then the 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, and 23F
  • CTAB cetyltrimethylammonium bromide
  • CTAB cetyltrimethylammonium bromide
  • the three serotypes 7F, 14, and 33F that do not react with CTAB was purified using an aluminum phosphate gel (Algel) solution.
  • the reaction with CTAB may comprise, for example, CTAB at a concentration of 1-20% (w / v) or 5-15% (w / v) in the reactant in an amount of 0.5-5% or 1-3% by weight (eg, 2 to 3% by weight for the polysaccharide derived from 23F, 1 to 2% by weight for the serotype-derived polysaccharide other than 23F) may be performed by addition, but is not limited thereto.
  • any one or more of pellet recovery after centrifugation, resuspension of the pellet with sodium chloride solution (eg, about 100-500 mM), and removal of CTAB ions with sodium iodide may be further performed. May be, but is not limited thereto.
  • the aluminum phosphate gel reaction may be performed by adding an aluminum phosphate gel solution to the reactants in an amount of 1 to 20 wt% or 5 to 15 wt%, but is not limited thereto.
  • the conjugate protein and the capsular polysaccharide are conjugated. It was prepared and used.
  • carrier protein refers to a protein that can be covalently conjugated with the capsular polysaccharide to increase the immunogenicity of the polysaccharide antigen. Specific types thereof are as described above. In one embodiment CRM197 can be used.
  • the carrier protein may be conjugated with the capsular polysaccharide through a standard conjugation method, and the capsular polysaccharide-carrying protein conjugate formed therefrom may be one or a plurality of capsular polysaccharides conjugated to one carrier protein.
  • All known methods for preparing conjugates of capsular polysaccharides and carrier proteins can be included within the scope of the present invention, wherein the conjugates are linked to the -OC (NH) -NH- group by using the cyanylation method.
  • the cyanation method may be appropriately performed by those skilled in the art through known methods, for example, may be performed using CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr, but is not limited thereto.
  • purified capsular polysaccharides can be chemically activated and each chemically activated capsular polysaccharide can be conjugated to the carrier protein one by one to form a glycoconjugate.
  • Cyanation activity by CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) treatment transforms the hydroxy group of the capsular polysaccharide into a cyanate group, which can be used to form a covalent bond with the amino group of the carrier protein CRM197. Can be.
  • the cyanation reaction by the CDAP may be specifically terminated by adding 3 molar equivalents of glycine (glycine) solution to 1 molar equivalent of CDAP and adjusting the pH to 9.0, but is not limited thereto.
  • the reaction solution and reaction conditions can be adjusted accordingly.
  • the capsular polysaccharide-carrying protein conjugates obtained can be purified by various methods. Examples of these methods include concentration / dialysis filtration processes, column chromatography and multilayer filtration. Purified polysaccharide-protein conjugates can be mixed and formulated into vaccine compositions of the invention and used respectively. Formulations of vaccine compositions of the invention can be carried out using methods known in the art. For example, 13 individual capsular polysaccharide-carrying protein conjugates can be formulated with a physiologically acceptable vehicle to make a composition. Examples of such vehicles may include, but are not limited to, water, buffered saline, water for injection, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or dextrose solutions.
  • a physiologically acceptable vehicle may include, but are not limited to, water, buffered saline, water for injection, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycols) or de
  • the term "vaccine” refers to a biological agent containing an antigen that immunizes a living body, and refers to an immunogen or antigenic substance that immunizes the living body by administering it to a human or an animal to prevent infection.
  • the vaccine composition may further include one or more selected from the group consisting of an adjuvant, a preservative, a buffer, a cryoprotectant, a salt, a divalent cation, a nonionic detergent, and a free radical oxidation inhibitor.
  • adjuvant in the present invention refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention.
  • the adjuvant is often provided to enhance the immune response, which is well known to those skilled in the art.
  • adjuvants suitable for increasing the effectiveness of the vaccine composition of the present invention may include, but are not limited to, one or more selected from the group consisting of:
  • aluminum salts eg, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.
  • oil-in-water emulsion formulations with or without muramyl peptide (defined below) or other specific immune stimulating agents, such as bacterial cell wall components
  • MF59 WO 90/14837
  • SAF 10% (w / v ) Squalene, containing 0.4% (w / v) tween 80, 5% (w / v) pluronic-block polymer L121 and thr-MDP (see below), and microfluidized with submicron emulsion ( microfluidization) or vortex to form a large particle size
  • saponin adjuvant such as Quill A or STIMULON® QS-21 (Antigenics, Framingham, MA, US Pat. No. 5,057,540), such as ISCOM ( Immunostimulatory complexes));
  • bacterial lipopolysaccharides synthetic lipid A homologues (eg, aminoalkyl glucoseamine phosphate compounds (AGP)), or derivatives or homologues thereof (commercially available from Corixa and described in US Pat. No. 6,113,918;
  • An example of is 2-[(R) -3-tetradecanoyloxytetradecanoylamino] ethyl 2-deoxy-4-O-phosphono-3-O-[(R) -3-tetradecanoyloxy Tetradecanoyl] -2-[(R) -3-tetradecanoyloxytetradecanoylamino] -bD-glucopyranoside, which is also known as 529 (formerly also known as RC529), which is aqueous or Formulated as a stable emulsion);
  • synthetic lipid A homologues eg, aminoalkyl glucoseamine phosphate compounds (AGP)
  • AGP aminoalkyl glucoseamine phosphate compounds
  • cytokines such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.) Interferon (eg gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and the like;
  • interleukins eg, interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL 12, IL-15, IL-18, etc.)
  • Interferon eg gamma interferon
  • GM-CSF granulocyte macrophage colony stimulating factor
  • MCSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • costimulatory molecules B7-1 and B7-2 costimulatory molecules
  • -2002/098368 and WO-2002 / 098369 pertussis toxin (PT), or E. coli heat-labile toxin (LT), in particular LT-K63, LT-R72, CT-S109, PTK9 / G129
  • WO Detoxified mutants of bacterial ADP-ribosylated toxins such as -93/13302 and WO-92 / 19265;
  • the muramyl peptides include N-acetyl-muramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1'-2 'dipalmityl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like, but is not limited thereto.
  • the aluminum salt adjuvant may be an aluminum-precipitated vaccine or an aluminum-adsorbed vaccine.
  • Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, amphogel, aluminum hydroxide, aluminum hydroxyphosphate adjuvant (APA), Amorphous alumina, and the like, but is not limited thereto.
  • APA refers to a suspension of aluminum hydroxyphosphate.
  • Aluminum chloride and sodium phosphate are mixed in a ratio of 1: 1 (by volume), aluminum hydroxyphosphate sulfate is precipitated, and the precipitate is set to 2 to 8 ⁇ m using a high shear mixer, and then dialyzed with physiological saline solution. It can be prepared by sterilization.
  • commercially available Al (OH) 3 eg alhydrogel or Superfos
  • Al (OH) 3 eg alhydrogel or Superfos
  • 50 to 200 g of protein can be adsorbed per mg of aluminum hydroxide, and this ratio is dependent on the protein's pi and the pH of the solvent. Low pI proteins bind more strongly than proteins with high pi.
  • Aluminum salts can form antigen reservoirs that slowly release antigens for two to three weeks to nonspecifically activate macrophages, complement, and innate immune mechanisms.
  • preservative means an anti-viral and / or antimicrobial agent that inhibits the growth of microorganisms in the vaccine composition, for example, chimerosal, phenoxyethanol ( 2-phenoxyethanol), formaldehyde, or mixtures thereof, but any conventional preservatives used in the art may be used.
  • the vaccine composition may comprise one or more physiologically acceptable buffers.
  • the buffer may have buffering capacity at pH 4.0 to 10.0, specifically, pH 5.0 to 9.0, more specifically pH 6.0 to 8.0.
  • the buffer may be at least one selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanolamine buffer. .
  • the buffer may be selected from buffers suitable for USP.
  • buffers include monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid; Dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid and phosphoric acid; It may be one or more selected from the group consisting of bases such as ammonia, diethanolamine, glycine, triethanolamine, TRIS.
  • the vaccine composition of the present invention may include a nonionic detergent.
  • a nonionic detergent for example, polysorbate 20 and polysorbate 80 in polyoxyethylene sorbitan esters (commonly called Tweens); Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (eg DOWFAX TM); Oxoxynols having a different repeating number of oxy-1,2-ethanediyl groups, especially ostoxynol-9 (Triton-100); Ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Nonylphenol ethoxylates such as NP series; Polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, oleyl alcohols, in particular triethyleneglycol monolauryl ether (Brij 30); Sorbitan ethers, also known
  • Tween 80 may be included in the emulsion and a mixture of nonionic detergents such as Tween 80 / Span 85 may be used. Combinations of polyoxyethylene sorbitan esters such as Tween 80 with octocinols such as Triton X-100 are also suitable, and combinations of Laureth 9 with Tween and or octosinol are also useful. Specifically, polyoxyethylene sorbitan esters such as Tween 80 may be used in an amount ranging from 0.01% (w / v) to 1% (w / v), in particular 0.1% (w / v); Octylphenoxy polyoxyethanol or nonylphenoxy polyoxyethanol (e.g.
  • Triton X-100 may range from 0.001% (w / v) to 0.1% (w / v), in particular from 0.005% (w / v) to 0.02% ( w / v);
  • Polyoxyethylene ethers e.g. laureth 9 are 0.1% (w / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v), in particular 0.1% (w / v) to 1% (w / v) or about 0.5% (w / v).
  • the vaccine composition of the present invention may be formulated in the form of a single dose dose vial, multiple dose dose vial or prefilled syringe.
  • a vial, or pre-filled syringe comprising the above-described vaccine composition in a single dose or in multiple doses, such as more than one dose.
  • the vaccine composition may further comprise a physiologically acceptable carrier.
  • a "multidose dose” or “multidose” means a vaccine dose capable of more than one dose (inoculation) to one administration (inoculation) or one or one dose of two or more administrations (inoculation). It can mean a vaccine dose that can be administered more than once (inoculation).
  • Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, oils.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate.
  • Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, buffer solutions.
  • oils are vegetable or animal oils, peanut oils, soybean oils, olive oils, sunflower oils, synthetic oils such as cod liver oil, marine oils, and lipids derived from milk or eggs.
  • Vaccine compositions of the present invention may be isotonic, hypertonic or hypotonic, and pharmaceutical compositions administered by infusion or injection are preferably but is not limited to isotonicity. On the other hand, isotonicity or hypertension may be advantageous for storage of the composition. If the vaccine composition is hypertonic, it can be diluted to isotonic prior to administration. Isotonic agents for dilution can be ionic tonicity agents such as salts or nonionic tonicity agents such as carbohydrates. Ionic isotonic agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, and the like. Nonionic isotonic agents include, but are not limited to, sorbitol, glycerol, and the like.
  • the amount of the conjugate at each vaccine dose may be chosen to be an amount that induces an immunoprotective response without significant side effects, which amount may vary depending on the serotype of pneumococcal.
  • the vaccine composition relative to the weight of the capsular polysaccharide derived from serotype 1 (ie, relative to 1 part by weight), serotypes 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A Weight ratios of 0.8 to 1.2 or 0.9 to 1.1 (ie 0.8 to 1.2 parts by weight or 0.9 to 1.1, respectively) for capsular polysaccharides derived from, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, respectively.
  • a weight ratio of 1.6 to 2.4, 1.8 to 2.2, or 1.9 to 2.1 ie, 1.6 to 2.4 parts by weight, 1.8 to 2.2 parts by weight, or 1.9 to 2.1 parts by weight. It may include, but is not limited to.
  • each conjugate may comprise 0.1 to 100 ⁇ g, specifically 0.1 to 10 ⁇ g, more specifically 1 to 5 ⁇ g polysaccharide.
  • the capsular polysaccharide derived from serotype 6B is identical to the capsular polysaccharide derived from serotype 6B.
  • the capsular polysaccharides derived from, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F are each in the amount of 2 to 2.4 ⁇ g or 2.1 to 2.3 ⁇ g, such as about 2.2 ⁇ g, respectively. 4 to 4.8 ⁇ g, 4.2 to 4.6 ⁇ g, or 4.3 to 4.5 ⁇ g, such as about 4.4 ⁇ g, but is not limited thereto.
  • CRM197 protein in the composition may be included in an amount of 0.1 to 100 ⁇ g, 1 to 50 ⁇ g, 20 to 40 ⁇ g, or 28 to 31 ⁇ g, such as about 29.3 ⁇ g, but is not limited thereto.
  • Optimal amounts of ingredients for a particular vaccine can be identified by standard studies involving the observation of an appropriate immune response in a subject. For example, the results of animal experiments can be extrapolated to determine the vaccination dose for humans. In addition, one skilled in the art can empirically determine its dose as needed.
  • the vaccine composition may further include aluminum element and sodium chloride, but is not limited thereto.
  • the vaccine composition according to the present invention can be used to protect a subject susceptible to pneumococcal and to prevent pneumococcal disease by administering a pharmaceutically effective amount in a systemic or mucosal route.
  • prevention of the present invention refers to any action that inhibits or delays infection by the pneumococcal by administration of the vaccine composition of the present invention.
  • a "pharmaceutically effective amount” refers to a dosage required to elicit an antibody that is capable of significantly reducing the probability of infection or the severity of infection.
  • administration of the present invention refers to the introduction of certain substances into an individual in any suitable way.
  • the vaccine composition of the present invention may be administered by inhalation route through oral, nasal, rectal, transdermal or aerosol, but may be administered by bolus or infused slowly, but is not limited thereto.
  • the administration may be by injection via an intramuscular, intraperitoneal, intradermal or subcutaneous route; Or mucosal administration to the oral / digestive tract, airway or urogenital tract, and the like.
  • intranasal administration can be used for the treatment of pneumonia or otitis media, in which case more effective prevention of nasopharyngeal carriers of pneumococci can attenuate the infection at an early stage.
  • the “subject” to which the vaccine composition or pharmaceutical composition of the present invention is administered may mean a living organism to which a pathogen may be infected or a cell, tissue or culture thereof separated from the organism, which organism may be a higher vertebrate. And, more specifically, it may be a mammal such as a human, but is not particularly limited thereto.
  • composition of the present invention may be administered in a single inoculation, or two, three, four or more times at appropriate intervals, but is not limited thereto.
  • routine inoculation plans for infants and newborns for invasive diseases caused by Streptococcus pneumoniae can be 2, 4, 6 and 12 to 15 months of age.
  • composition may further comprise one or more proteins from Streptococcus pneumoniae.
  • Streptococcus pneumoniae proteins suitable for inclusion may include all of the proteins identified in WO-2002 / 053855, as well as the proteins described in WO-2002 / 053761, within the scope of the present invention.
  • each polysaccharide except that 6B-derived polysaccharide, is 4.4 ⁇ g in a total of 0.5 mL of the vaccine composition; About 29.3 ⁇ g CRM197 transport protein; 0.5 mg of elemental aluminum (2 mg aluminum phosphate) adjuvant; About 4.25 mg sodium chloride (without preservatives) or about 3.5 mg (with preservatives); About 295 ⁇ g succinate buffer; And about 3 mg of 2-phenoxyethanol and about 60 ⁇ g of formaldehyde, such as 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24 A pvalent, 14 to 19, 14 to 17, or 14 to 15 pneumococcal vaccine composition is exemplified.
  • serum levels of rabbits inoculated with the vaccine composition confirmed higher serotype specific IgG concentrations than Prevena 13® (Table 1).
  • the functional immunogenicity test (Opsonophagocytic assay) showed a superior effect than the prebena 13 ® (Table 2).
  • Another embodiment of the present invention is capsular polysaccharide-carrying protein conjugate 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19 It is an immunogenic composition for pneumococci, including species, 14 to 17 species, or 14 to 15 species.
  • the conjugate and pneumococcus are as described above.
  • the composition comprising 14 to 15 species is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 to 17 species of the capsular polysaccharide-carrying protein conjugate of the present invention
  • the composition comprising 14 to 15 species is 13 to 24, 13 to 19, 13 to 17, 13 to 15, 14 to 24, 14 to 19, 14 To 17, or 14 to 15 different serotypes include streptococcus pneumoniae-derived capsular polysaccharides, and when administered in vivo to recognize the antigen to produce antibodies It causes an immune response and can be used as an immunogenic composition against pneumococci.
  • Another aspect of the invention is a method of preventing pneumococcal disease by administering the vaccine composition or immunogenic composition to a subject in need thereof.
  • Another aspect of the invention includes 13 capsular polysaccharide-carrier protein conjugates, wherein the 13 conjugates are Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, Each of 13 capsular polysaccharides derived from 9V, 14, 18C, 19A, 19F and 23F is covalently conjugated to a carrier protein, the carrier protein is CRM197 protein, and the conjugate is a cyanylation method.
  • the composition has a structure in which the capsular polysaccharide and the carrier protein are linked by -OC (NH) -NH- groups.
  • Vaccine compositions immunogenic compositions, and prevention of pneumococcal disease are as described above.
  • Capsular polysaccharide is a method for producing the immunogenic composition comprising the step of conjugating each of the capsular polysaccharide and the carrier protein CRM197 to have a structure linked by -OC (NH) -NH- group using a cyanylation method.
  • the multivalent pneumococcal vaccine composition according to the present invention comprises a multivalent conjugate of capsular polysaccharide-protein of unique conjugation structure, and includes an optimized combination and content of 2-phenoxyethanol and formaldehyde as a preservative, thereby providing excellent immunogenicity. It is characterized by excellent stability and / or preservation while maintaining. Therefore, the vaccine composition and the immunogenic composition according to the present invention can be used more safely and usefully in preventing diseases caused by pneumococci in infants, infants, children, and adults.
  • Streptococcus pneumoniae with 16 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B) Obtained from the US Center for Disease Control and Prevention (CDC), cell banks were prepared in the following manner.
  • Streptococcus pneumoniae strains were smeared on blood agar medium to identify pneumococci and the existing media components were removed. After growing a single colony out of 10 or more single colonies, inoculated and cultured in liquid medium (Soytone (Kerry Bio-Science) or Yeast extract (Bio-springer) -derived medium) containing no animal-derived ingredients After the addition, synthetic glycerol was added to prepare a research cell bank (RCB) containing synthetic glycerol.
  • RBC research cell bank
  • Master cell banks were prepared by taking out one vial from the cell bank for which the expression of polysaccharides with unique serotypes was confirmed, proliferating the cells in a liquid medium containing no animal-derived components, and adding synthetic glycerol. One vial was taken out of the bank to proliferate cells in a liquid medium containing no animal-derived components, and then synthetic glycerol was added to prepare a cell bank for production.
  • the prepared cell banks were stored in an ultra-frozen state of -70 deg.
  • the culture was carried out while maintaining the pH of the medium at 7.2 ⁇ 0.2 using a potassium hydroxide solution sterilized with minimal stirring at 37 ⁇ 2 ° C. Sampling was carried out after 2 hours from the start of the culture to measure the cell concentration in the culture medium and the glucose concentration in the medium. The culture was terminated when glucose in the medium was depleted.
  • CTAB cetyltrimethylammonium bromide
  • NaCl sodium chloride
  • NaI sodium iodide
  • the reaction was performed by adding an aluminum phosphate gel (Algel) solution, followed by treating 10% by weight of an aluminum phosphate gel solution with respect to the total process solution 1 The reaction was time. The supernatant obtained through centrifugation (1 l centrifugation at 17,000 ⁇ G conditions) was recovered and used for the subsequent process.
  • Algel aluminum phosphate gel
  • the sample was subjected to a depth filter and ultrafiltration (UF / DF), and then the amount of ethanol and sodium chloride was adjusted and stored in a raw form.
  • UF / DF depth filter and ultrafiltration
  • serotype 9V polysaccharide and CRM197 carrier protein GenBank Accession No. 1007216A; SEQ ID NO: 1; mgaddvvdssk sfvmenfssy hgtkpgyvds iqkgiqkpks gtqgnydddw kefystdnky daagysvdne nplsgkaggv vkvtypgltk vlalkvdnae tikkelglsl teplmeqvgt eefikrfgdg asrvvlslpf aegssveyi nnweqakals veleinfetr gkrgqdamye ymaqacagnr vrrsvgssls cinldwdvir dktktkiesl kehgpiknkm sespnkt
  • the equilibrated complex was dissolved by incubation (37 ⁇ 2 ° C.) in sodium phosphate (Na 3 PO 4 ) buffer solution at a rate of 0.1 M per 20 g of saccharides, and then cyanoborohydride (100 mg / mL) was added to the protein and The conjugation reaction between sugars was initiated. After incubation at 37 ⁇ 2 ° C. for about 44-52 hours, the temperature was lowered to 23 ⁇ 2 ° C. and 1 mL of 0.9% (w / v) NaCl solution was added to the reactor.
  • Sodium borohydride solution (100 mg / mL) was added so as to have 1.8 to 2.2 molar equivalent of sodium borohydride per mole of saccharide, and the reaction mixture was incubated with stirring at 23 ⁇ 2 ° C. to be present in saccharide. Any aldehydes that did not react were reduced. 5 mL of 0.9% (w / v) aqueous sodium chloride solution was added to the obtained saccharide-protein conjugate mixture, and the diluted conjugate mixture was dialyzed and filtered using a 100 kDa MWCO membrane.
  • a 2M NaCl polysaccharide solution was prepared by adding sodium chloride powder to a serotype 9V polysaccharide stock solution prepared without hydrolysis.
  • a CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) solution was added to the serotype 9V polysaccharide solution to a concentration of 0.5% (w / w) relative to the polysaccharides, followed by stirring for 15 minutes to perform the polysaccharide activation reaction. Induced.
  • CRM197 was added to the polysaccharide solution after the polysaccharide activation process so that the ratio of CRM197 to polysaccharide was 1.0% (w / w) (CRM197 weight / polysaccharide weight), and the conjugation reaction was performed at room temperature for 1 hour.
  • the conjugation reaction was terminated by adding 2M glycine solution in 3 molar equivalents to 1 molar equivalent of CDAP and incubating overnight at room temperature with pH adjusted to 9.0.
  • the terminated conjugate was concentrated and diafiltered into an ultrafiltration filter through a buffer containing 0.9% (w / w) sodium chloride.
  • the original capsular polysaccharide derived from each serotype was dissolved in water for injection, respectively, so that the final concentration range was within the range described below and filtered through a 0.45 ⁇ m filter:
  • serotypes 6A, 12F and 19A range from 8 to 12 mg / ml
  • the hydrolysis was then stopped by cooling to 21 ° C.-24 ° C. and adding sodium hydroxide to a target pH of 6.0 ⁇ 1.0.
  • CDAP 2 (w / w) versus polysaccharide for serotypes 2 and 4,
  • CDAP 3 (w / w) versus polysaccharide for serotypes 1, 3, 7F, 15B, 19F and 19A
  • CDAP 4 (w / w) versus polysaccharide for serotypes 5, 6B, 18C, and 23F;
  • Serotype 12F was dissolved at a CDAP 5 (w / w) to polysaccharide ratio and added to each polysaccharide solution.
  • CRM197 was added to each serotype polysaccharide solution in an amount of CRM197 0.75 (w / w) relative to the polysaccharide, followed by conjugation reaction for 2 hours. Thereafter, the reaction conversion was measured using SE-HPLC, and additional CDAP was added as necessary.
  • the diluted conjugation mixture was concentrated and diafiltered in an ultrafiltration filter using a minimum of 20 volumes of buffer (5 mM Succinate buffer, pH 5.8 with 150 mM NaCl).
  • a buffer containing 0.9% (w / v) sodium chloride was used while maintaining a pH range of 5.5 to 6.5 as a buffer.
  • Fractional molecular weight of the ultrafiltration filter was carried out using 300 kDa in all serotypes, and the permeate was discarded.
  • the residue after diafiltration was diluted to less than 0.4 g / L based on polysaccharide content concentration using a buffer (5 mM Succinate buffer (pH 5.8) with 150 mM NaCl) and filtered through a 0.22 ⁇ m filter.
  • the filtered product was subjected to in-process control (sugar content, residual DMAP). In-process controls were performed on the filtered residue to determine if further concentration, diafiltration and / or dilution were needed.
  • Aluminum salt (aluminum phosphate) was added to the sterile filtrate so that the final concentration was 1 mg / mL based on aluminum ions, and an additional salt was added to maintain a pH range of 5.5 to 6.5. After the adsorption, the stock solution was subjected to quality inspection to confirm quality suitability, and refrigerated at 2 to 8 ° C. until use.
  • Polyvalent pneumococcal polysaccharide-protein conjugates were prepared from the capsular polysaccharide-protein conjugates combined with and CRM197 protein as follows:
  • the required amount of conjugate bulk solution was calculated based on the batch volume and the polysaccharide concentration of the conjugate stock solution.
  • Step 6 of Preparation Example 2 to the conjugate bulk solution of each serotype and to the addition of aluminum phosphate, 0.85% (w / v) saline and 5 mM succinate buffer (pH 5.8) were added, Bulk solutions of each conjugate were prepared containing 0.85% sodium chloride, 5 mM succinate buffer (pH 5.8) and 1 mg / mL elemental aluminum (concentration of elemental aluminum in aluminum phosphate).
  • chimerosal 85 or 100 ug / ml or 2-phenoxyethanol (2-PE) (0, 5.0, or 10.0 mg / ml) and formaldehyde (0, 10, 25, 40, 50, 80 , 100, 120, or 170 ug / ml) was added and slowly mixed into the serotype combinations above to prepare a formulated multivalent pneumococcal conjugate vaccine composition.
  • concentration of each serotype capsular polysaccharide of the multivalent vaccine composition is 4.4 ⁇ g / mL (where Type 6B is 8.8 ⁇ g / mL). pH was checked and adjusted to pH 5.8 if necessary.
  • the formulated final vaccine composition stock prepared above was filled into Type 1 borosilicate glass vials. The filled vaccine composition was stored at 2-8 ° C.
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F
  • 13 vaccine serum type 1, 3, 4, 5, 6
  • Prevenar 13® used as the vaccine composition LBVE013 or positive control, was administered in New Zealand White at parking 0, parking 2 and parking 4 at planned human clinical dose (2.2 ⁇ g of each polysaccharide, exception: 4.4 ⁇ g of 6B).
  • Rabbits were immunized into muscles and serum was taken at 2 week intervals after inoculation.
  • Table 1 shows the results of IgG measurement using ELISA on the collected serum. This will be described in detail as follows.
  • Capsular polysaccharides for each of the 13 serotypes were treated in 96-well plates at 5 ⁇ g / well and coated for 16 hours at room temperature.
  • serum of each individual was taken in the same amount and pooled between the same groups.
  • Serum pool was adsorbed with 333.3 ⁇ g / mL of C-PS (Cell Wall Polysaccharide; State Serum Institute) and 333.3 ⁇ g / mL of capsular polysaccharide (PnPs22F) of serotype 22F at room temperature for 30 minutes.
  • the serotype-specific immunogenic response pattern of the multivalent (13-valent) pneumococcal vaccine composition is significantly different from that of Prevena 13®.
  • a higher level of titer than prevena 13® was observed in all serotypes compared to prevena 13® in rabbits inoculated with the trivalent vaccine composition (LBVE013) according to this example, in particular, serotypes 1, 6B, 7F, and 9V. At 14 and 19F, they were two to six times better than Prevena 13®.
  • OPA analysis was performed on sera from rabbits to evaluate the function of antibodies induced by serotypes.
  • THY medium Todd-Hewitt Broth w / 2% Yeast Extract
  • Phagocytosis was stopped by lowering the temperature, and 10 ⁇ l of the reaction solution was plated in dried agar medium for 30 to 60 minutes in advance. Next, the cells were incubated for 12 to 18 hours in a CO 2 incubator (37 ° C.) and the number of colonies was counted. OPA titers were expressed as dilution multiples where 50% killing was observed.
  • the OPA titer indicated as 2187 means that the titer is very high even when the most diluted section does not reach the 50% level compared to the negative control.
  • the multivalent pneumococcal vaccine composition according to the present example had a significantly superior serum IgG titer compared to Prevena 13®. Therefore, the multivalent pneumococcal vaccine composition according to the present embodiment can be very useful for preventing diseases caused by pneumococcal.
  • the antiseptic test of the vaccine was conducted according to the EP-B standard, which is a standard required for vaccine products by the World Health Organization (WHO) among the US Pharmacopoeia (USP) and the European Pharmacopoeia (EP).
  • WHO World Health Organization
  • USP US Pharmacopoeia
  • EP European Pharmacopoeia
  • the two bacteria bacteria Pseudomonas aeruginosa Four species of (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), Yeast Candida albicans (ATCC No. 10231, CA), and Fungal Aspergillus niger (ATCC No. 16404, AN) were added to the vaccine composition. Hours were inoculated with 10 5 to 10 6 CFU / mL (CFU; Colony forming units). Thereafter, the samples were sampled at 24 hours, 7 days, 14 days and 28 days, cultured in a solid medium, and the number of colonies was counted at 3 to 5 days.
  • CFU Colony forming units
  • Table 3 shows the antiseptic properties of EP-B and the pharmacopoeia of the countries according to the method.
  • EP requirements are more stringent than the USP or USP, and are classified into categories A and B according to the formulation, and the level required by the WHO for vaccine products is EP-B (EP 5.1.3.Efficacy of antimicrobial perserbation, USP 37-51, Antimicrobial effectiveness testing).
  • chimerosal and 2-PE which are vaccine preservatives commonly used in the art, were added and subjected to an antiseptic test, and chimerosal or a low concentration of 2-PE. When used alone it was confirmed that does not satisfy the antiseptic force criteria.
  • the inventors of the present invention experiment to develop a new composition that can increase the antiseptic effect while minimizing the content of 2-PE in order to satisfy the antiseptic standard without infringing patents of other companies as a preservative of the multivalent pneumococcal protein conjugate vaccine. As a result, the following results were obtained.
  • the multivalent pneumococcal protein conjugate of vaccine compositions 1 to 12 was added by adding chimerosal, or 2-PE and / or formaldehyde, as shown in Table 2 below.
  • Antiseptic test was carried out according to the test method of EP-B.
  • the preservative of the multivalent pneumococcal polysaccharide-protein conjugated vaccine contains 2-PE in the amount of about 5 mg / mL and formaldehyde in the amount of about 100 ⁇ g / mL, it was confirmed that the antiseptic capacity of EP-B standard was satisfied. .
  • the pneumococcal protein conjugate vaccine typically uses 2-PE 7 mg / mL or more as a preservative, and together with the result of Example 3, the result shows that when formaldehyde is added in an amount of 100 ⁇ g / mL or more, Lowering the -PE content to 5 mg / mL shows that the antiseptic power of EP-B can be achieved.
  • the present specification provides a strong and effective antiseptic force by applying an optimized combination of 2-phenoxyethanol (2-PE) and formaldehyde (HCHO) as a preservative of the multivalent pneumococcal polysaccharide-protein conjugate vaccine. It has become possible to provide a multidose composition to maintain.
  • vaccine composition 16 was prepared by the following method: 14 polysaccharide-protein conjugates (Type 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) ) And 1 mg / mL of aluminum phosphate (based on aluminum concentration) were stirred to mix well. 5 mM succinic acid and 0.85% (w / v) sodium chloride were dissolved in distilled water for injection, followed by mixing with a sterile filtered buffer.
  • the combination of 2-PE and formaldehyde as exemplified in the examples of the present invention is an excellent composition capable of effectively maintaining the antiseptic power of the multivalent pneumococcal polysaccharide-protein conjugated vaccine for a long time, and the polyvalent polysaccharide-protein conjugate It has been found that the vaccine can be usefully used to provide multidose compositions with improved antiseptic or extended antiseptic retention periods.

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Abstract

La présente invention concerne une composition vaccinale contre Streptococcus pneumoniae et, plus particulièrement, une composition vaccinale comprenant : (i) un conjugué polysaccharide-protéine capsulaire; (ii) 2-phénoxyéthanol (2-PE); et (iii) formaldéhyde (HCHO), et son procédé de préparation.
PCT/KR2018/002983 2017-03-15 2018-03-14 Composition vaccinale polyvalente contre streptococcus pneumoniae WO2018169303A1 (fr)

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US11116828B2 (en) 2017-12-06 2021-09-14 Merck Sharp & Dohme Corp. Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
CN114544841A (zh) * 2020-11-24 2022-05-27 湘潭智联技术转移促进有限责任公司 一种采用高效液相色谱法测定肺炎球菌多糖-蛋白结合疫苗中dmap残留量的方法
US11642406B2 (en) 2018-12-19 2023-05-09 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
US12016914B2 (en) 2023-03-23 2024-06-25 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

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TW202116352A (zh) * 2019-07-18 2021-05-01 南韓商賽特瑞恩股份有限公司 包含多價肺炎球菌多醣-蛋白共軛物的免疫組成物、醫藥組成物及其用途
WO2022031087A1 (fr) * 2020-08-07 2022-02-10 (주)셀트리온 Composition immunogène comprenant des conjugués polysaccharide pneumococcique-substance dérivée de paroi de cellulaire
KR20220102871A (ko) * 2021-01-14 2022-07-21 (주)셀트리온 다가 폐렴구균 다당류-단백질 접합체를 포함하는 면역원성 조성물
CN113173977B (zh) * 2021-05-12 2024-03-29 江苏坤力生物制药有限责任公司 一种双功能抗原、其制备方法及应用

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US20190070282A1 (en) * 2015-07-21 2019-03-07 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof
US11020469B2 (en) * 2015-07-21 2021-06-01 Pfizer Inc. Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof
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US11642406B2 (en) 2018-12-19 2023-05-09 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof
CN114544841A (zh) * 2020-11-24 2022-05-27 湘潭智联技术转移促进有限责任公司 一种采用高效液相色谱法测定肺炎球菌多糖-蛋白结合疫苗中dmap残留量的方法
US12016914B2 (en) 2023-03-23 2024-06-25 Merck Sharp & Dohme Llc Compositions comprising Streptococcus pneumoniae polysaccharide-protein conjugates and methods of use thereof

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