The invention pneumococcus (Streptococcus pneumoniae; Streptococcus pneumoniae ) relates to a vaccine composition, more specifically, (i) capsular polysaccharides (capsular polysaccharide) - protein conjugate, (ii) 2- phenoxyethanol (2-Phenoxyethanol , to a 2-PE), and (iii) formaldehyde (vaccine composition and a method for manufacturing the same including a formaldehyde, HCHO).
BACKGROUND
[2]
Pneumococcus (Streptococcus pneumoniae ( Streptococcus pneumonia ); pneumococcus) is the main cause of a type of streptococcus showing gram-positive and hemolytic, worldwide meningitis in infants, children and the elderly, pneumonia, and severe invasive infections. And more than 1.6 million people die each year from disease caused by Streptococcus pneumoniae, and the incidence of (2008, World Health Organization data), especially in the immune abilities of invasive infections caused by Streptococcus pneumoniae in older age groups than low 5 years of age children and 65 years of age high.
[3]
Streptococcus pneumoniae has been classified into 90 kinds of more than serotype (serotype) according to the structural and immunological properties of the major virulence factors of capsular polysaccharide (virulence factor) that surrounds the outside (cell membrane), a double 20 of the in humans it is known that there are 80 to 90% associated with virulence. The only host of Streptococcus pneumoniae are human, they are usually present in a community built on the nasopharynx (nasopharynx) of healthy individuals (20 to 40 percent of children 5 to 10% of adults). 2005 US Centers for Disease Control and Prevention (Centers for Disease Control and Prevention; CDC) is that about 210 million five deaths of more than children are by pneumonia three and the only one of 120 people only a year in developing countries pneumococcal deaths by infection were reported, the meningitis and septicemia caused by pneumococci were counted as occurring about 3,000 cases per year and 50,000 cases respectively within the United States (Peters TR, Poehling KA et al Invasive pneumococcaldisease JAMA 2007; 297:.. 1825-6). In addition, according to the aggregation of Streptococcus pneumoniae pneumoACTION can be called a data base of bottles, children in the country in 2000, pneumococcal infections was 24,047 cases were caused, the death of 47 cases per year (www.pneumoadip.org). Furthermore, a recent Centers for Disease Control and Prevention has announced According to the study on the pneumococcal serotype analysis of domestic Pediatrics' Streptococcus pneumoniae in infants of between 3-59 months to be the most common (43.7%) cause of invasive infections appear.
[4]
Thus, pneumococcal a situation where the need for pneumococcal vaccination for children and the elderly for the risk of infection is constantly raised.
[5]
In order to prevent pneumococcal infections, multivalent pneumococcal polysaccharide vaccines in development since 1977, been approved, these capsular polysaccharide vaccine has been proven to be useful in the prevention of pneumococcal disease in elderly and high-risk patients. However, in the case of children there since the maturity of the immune system so if a fall compared to adults only hit polysaccharide vaccine, the immune system does not recognize the polysaccharide antigen to external factors difficult to break the expected role as a vaccine. Thus handling which increases the immunogenicity to the polysaccharide antigen to correct the decrease immunogenicity of polysaccharide vaccines problem in infants and children with a protein (carrier protein) bonding the capsular polysaccharide-protein conjugate vaccine in 7 the pneumococcal conjugate vaccine ( 7vPnC, Prevenar ® (Prevenar®)) were used have been developed, and has been reported to be effective for the prevention of invasive disease and otitis media in infants and children in many materials. However, the use of the 7 a vaccine, but leads to a reduction of invasive disease due to the vaccine serotypes used in the vaccine In addition showed a relative increase pneumococcal disease caused by some non-vaccine serotypes. Thus, 10 the capsular polysaccharide-protein conjugate vaccine in renal flow Rix ® (Synflorix®) and Prevenar ® basic serotype 13 is added to a pneumococcal conjugate vaccine Prevenar 13® (Prevenar13® a serotype 6 species of ) have been developed, but presently on the market, in situations where possible have been reported that the efficacy as a vaccine may be insufficient [Andrews NJ et al, (2014) for containing some serotypes Lancet Infec Dis (14) 839;
[6]
On the other hand, a vaccine dose injections are to be use preservatives in order to avoid contamination of microorganisms. Through the UN, including vaccine products to be exported to developing countries is a multi-dose administration of the product due to the use of the national environment, distribution methods, and costs, including preservatives are preferred. As a preservative for use in vaccine products and the like chimerosal (Thimerosal), phenoxyethanol (2-Phenoxyethanol, 2-PE), phenol (Phenol) the amount of preservative commonly used in the art. For example chimerosal is a 10 ㎍ / mL concentration of, 2-PE is 5 mg / used in mL concentration of and, multidose amount of the vaccine product that contains this is EP-B (European Pharmacopoeia B category) or the USP (United States Pharmacopoeia) can be commercialized must pass the tests of buoyancy room standards.
[7]
Chimerosal (Thimerosal, Thiomersal, merthiolate) is ethyl mercury derivative has been used as a preservative in multidose amount of vaccine injection since the early 1930's. Chimerosal prevent the growth of contaminated microorganisms vaccine products in storage or in use, and has been used for the purpose of maintaining a sterile WHO PQ (Prequalified) a number of 5 is a liquid combination vaccine (D, T, P, Hib obtaining , including HBsAg) may contain chimerosal as preservatives.
[8]
2-Phenoxyethanol (2-PE) is mainly used as a preservative in cosmetics, transdermal-type drug and has been used as a preservative in the vaccine injections.
[9]
However, in these antiseptic ingredients can lead to the amount toxicity and / or side effects that are used to achieve enough room buoyancy of the vaccine, it is necessary to develop a technique for reducing give enough room buoyancy their usage.
Detailed Description of the Invention
SUMMARY
[10]
One example is (i) Streptococcus pneumoniae ( Streptococcus pneumoniae capsular polysaccharide) (capsular polysaccharide) - protein conjugate, (ii) 2- phenoxyethanol (2-Phenoxyethanol, 2-PE ), and (iii) formaldehyde Streptococcus pneumoniae provides a vaccine composition comprising a (Formaldehyde, HCHO). The vaccine composition may be a multidose amount of a vaccine composition for multidose.
[11]
Another example provides a pharmaceutical composition for preventing or treating a Streptococcus pneumoniae vaccine comprising the composition of Streptococcus pneumoniae infections. The pharmaceutical composition for the treatment or prevention may be a multidose amount of a pharmaceutical composition for multidose.
[12]
Other examples are Streptococcus pneumoniae ( Streptococcus pneumoniae ) of capsular polysaccharides (capsular polysaccharide) - protein preparing a conjugate, and wherein the capsular polysaccharide-protein conjugates, and 2-phenoxyethanol (2-Phenoxyethanol, 2-PE ) and formaldehyde (formaldehyde, HCHO), the comprising the step of mixing, stability and / or preservative (or room buoyancy), stability of the production process of an enhanced pneumoniae vaccine composition or a pneumococcal vaccine and / or a preservative (or room buoyant ) provides the enhancement method. The vaccine composition may be a multidose amount of a vaccine composition for multidose. The capsular polysaccharides comprising the steps of preparing a protein conjugate may comprise the step of connecting via a carbonylation cyano (cyanylation) to perform a method capsular polysaccharide and the protein carrying the -OC (NH) -NH-.
Problem solving means
[13]
One example is (i) Streptococcus pneumoniae ( Streptococcus pneumoniae capsular polysaccharide) - protein conjugate, (ii) 2- phenoxyethanol (2-Phenoxyethanol, 2-PE ), and (iii) formaldehyde (Formaldehyde, HCHO ) Streptococcus pneumoniae vaccine composition or a pneumococcal provides an immunogenic composition comprising a.
[14]
As used herein, Streptococcus pneumoniae immunogenic composition is to mean a composition for inducing an immune response against Streptococcus pneumoniae, is used as a, it means Streptococcus pneumoniae and the same vaccine composition, unless otherwise described.
[15]
Right standing described herein, may refer to "comprising (a given component)," is essentially a one (comprising) or described components, which may include components other than the described component (consisting essentially of) .
[16]
Pneumococcal vaccine compositions provided herein are Streptococcus pneumoniae ( Streptococcus pneumoniae and capsular polysaccharides (capsular polysaccharide) of a), by including 2-phenoxyethanol with formaldehyde as a preservative, inducing immune responses to pneumococcal efficacy (immunogenicity) can be held long-term stability and / or preservative (room buoyancy) the immunogenic composition is promoting.
[17]
The capsular polysaccharides (capsular polysaccharide) is her new moniker Streptococcus ( Streptococcus pneumoniae) It may be derived from the capsular polysaccharide. Specifically, the capsular polysaccharide is derived from at least two of Streptococcus pneumoniae serotypes, for example, more than five species, more than seven species, more than nine species, more than 11 species, more than 13 species, or more than 14 kinds of serotype It may be a capsular polysaccharide. In one embodiment, the capsular polysaccharide, Streptococcus pneumoniae serotype of, 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds of to 19 species, it can be to include capsular polysaccharides derived from 14 kinds to 17 kinds or 14 kinds to 15 kinds of serotypes. E.g., the capsular polysaccharide is Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A , 19F, 20, 22F, 23F, and 13 kinds to 24 member selected from the group consisting of 33F, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 species, it may be one containing the capsular polysaccharides derived from 14 species to 17 species, or 14 kinds to 15 kinds of serotypes. In one embodiment, the capsular polysaccharide is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and capsular 13 kinds derived from 23F polysaccharide; Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 14 kinds of capsular polysaccharides derived from 23F; Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 15 kinds of capsular polysaccharides derived from 23F, 2, and 12F; Streptococcus pneumoniae serotypes 1, 3, 4, 15 kinds of capsular polysaccharides derived from 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; Or Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, the 17 kinds of capsular polysaccharides derived from 19F, 22F, 23F and 33F It is to include, but is not limited to this.
[18]
Wherein the capsular polysaccharide-protein conjugates may be a capsular polysaccharide of serotype 2, derived from at least one kind described above, each individual protein to the conventional method, for example, through a covalent bond, bonded to each other. In one example wherein the conjugate is biotinylated cyano (cyanylation), each of the capsular polysaccharide using the method (specifically, the hydroxy group of the polysaccharide) and the protein structures are linked through a (more specifically, the amino group of the protein) is -OC (NH) -NH- may be one having a ([polysaccharide; -OC (NH) -NH- [protein]). When bonding the capsular polysaccharide and protein using cyano carbonylation method as described above, as compared with other methods (e. G. Reduction amination method and the like), to the induction degree of structural deformation of the capsular polysaccharide little, maintaining the immunogenicity after bonding It will be more advantageous. For example, a particular serotype (19F, etc.) are, in some cases in which the protein and bonded to a reducing amination method reaction combination hexasaccharide ring structure is it can also result in the cutting open structure that might occur if the one immunogenicity of this degradation On the other hand, when it is bonded to proteins and shea annealing method it has been reported that there is no possibility of these problems occurring.
[19]
The protein transport (carrier; carrier) may be be a protein and, for example, proteins that can be obtained in sufficient quantity and purity denotes a non-toxic and non-reactive. Threatening polysaccharides derived from different serotypes may be the same or different and bonded proteins, such as the same proteins, respectively.
[20]
In one embodiment, the protein may be a protein CRM197. CRM197 protein is Corynebacterium diphtheria strain C7; a non-toxic variant of diphtheria toxin isolated from cultures of (e.g. CRM197 casamino acids (casamino acid) and yeast extract growth base in the medium), that is, the modified toxin (toxoid). CRM197 protein is described in (incorporated herein by reference), ultrafiltration, ammonium sulfate precipitation and ionic exchange via chromatography or purified from Corynebacterium diphtheria strain C7 culture, conventional methods, for example, U.S. Patent No. 5,614,382 No. see the method may be obtained recombinantly. In one example, CRM197 protein, GenBank Accession No. It may be one comprising an amino acid sequence having an amino acid sequence or the sequence of 1007216A and at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or 99.9% or more homology.
[21]
Further, FIG modified toxin derived from Corynebacterium diphtheria strain C7 than diphtheria can be used as transport protein.
[22]
This is in addition to using available transport proteins
[23]
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 Escherichia coli ST, and exotoxin a of Pseudomonas Ke rugi industrial origin;
[24]
Bacterial outer membrane proteins such as, outer membrane complex c (OMPC), morpholine (porins), transferrin binding proteins, New Mora, who, pneumococcal surface protein A (PspA), pneumococcal adjuster hesin (adhesin) protein (PsaA), the group A or of group B streptococci C5a peptidase derived, to a brush Russ influenza (Haemophilus influenzae) protein D;
[25]
Ovalbumin, keyhole rimpet not H. linen (KLH), bovine serum albumin (BSA), a protein derivative (PPD) purified in tyubeo kyulrin (tuberculin);
[26]
Mutant (modified toxin) of diphtheria toxin such as CRM197, CRM173, CRM228, CRM45;
[27]
It may be at least one member selected from the group consisting of and the like.
[28]
Wherein the capsular polysaccharide-protein conjugates derived from at least two of Streptococcus pneumoniae serotypes, for example, more than five species, more than seven species, more than nine species, more than 11 species, more than 13 species, or more than 14 kinds of serotype polyhydric containing each capsular polysaccharide as described previously transport proteins, such as the CRM197 protein conjugate is bonded polysaccharide-protein conjugates may be. In one embodiment, the capsular polysaccharide-protein conjugates, Streptococcus pneumoniae serotypes of, 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds , the 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds, each capsular polysaccharide and carrier proteins, for example, CRM197 13, which protein comprises the bonding conjugate derived from serotype to 24, 13 a and 19, 13 is to 17 the, 13 a to 15 a, 14 a to 24 a, 14 a to 19 a, 14 a to 17, or 14 are to 15. the polysaccharide-may be a protein conjugate. E.g., the capsular polysaccharide-protein conjugate is Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 13 selected from the group consisting of 33F species to 24 species, 13 species to 19 species, 13 species to 17 species, 13 species to 15 species, 14 species to 24 species, 14 the species to 19 species, 14 species to 17 species, or 14 species to 15 species, each capsular polysaccharide and carrier proteins, for example, 13 to CRM197 protein is a conjugate comprising a joint derived from serotype to 24, 13 a and 19, 13 is to 17 the, 13 a to 15 a, 14 a to 24 a, 14 a to 19 a, 14 a to 17, or 14 are to 15. The polysaccharide-may be a protein conjugate. In one embodiment, 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 species of capsular polysaccharide and carrier protein, respectively, for example, the 13 polysaccharides comprising the conjugates of CRM197 protein is a 13 bonded species-protein conjugate; Streptococcus pneumoniae polysaccharide is 14 derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F - protein conjugate; Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and the 15 kinds of capsular polysaccharides derived from 12F, respectively, and transport proteins, for example, the 15 polysaccharides comprising the conjugates of CRM197 protein is a 15 bonded species-protein conjugate; Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and the 15 kinds of capsular polysaccharides derived from 15B respectively, and transport proteins, for example, the 15 polysaccharides comprising the conjugates of CRM197 protein is a 15 bonded species-protein conjugate; Or Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and each of the 17 kinds of capsular polysaccharides derived from 33F and transport proteins, for example,
[29]
Streptococcus pneumoniae vaccine composition provided herein is a capsular polysaccharide-pneumococcal conjugate vaccine comprising a protein conjugate; a (Pneumococcal conjugate vaccine PCV). The pneumococcal vaccine compositions, respectively derived from two or more kinds of the Streptococcus pneumoniae serotypes, for example, 5 or more, 7 or more, 9 or more species, more than 11 species, more than 13 species, or more than 14 kinds of serotype the capsular polysaccharide and the above-described transport proteins, for example, CRM197 protein is bonded polysaccharide-may be a multivalent pneumococcal vaccine composition comprising a protein conjugate. In one embodiment, the Streptococcus pneumoniae vaccine composition, Streptococcus pneumoniae of the cliff serotype, 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds of each capsular polysaccharide and carrier protein derived from a serotype of to 15 species, for example, CRM197 protein is conjugated 13 kinds to 24 kinds, 13 kinds to 19 kinds , 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds or 14 kinds to 15 kinds of polysaccharides - 13 comprising a protein conjugate to 24 is , is a 13 a to 19 a, 13 a to 17, and 13 are to 15, a 14 a to 24, be a 14 a to 19 a, 14 a to 17 a, and 14 a to 15 a Streptococcus pneumoniae vaccine composition . For example, the Streptococcus pneumoniae vaccine composition 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,
[30]
In one embodiment, the Streptococcus pneumoniae vaccine composition
[31]
(1) Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 13 kinds of capsular polysaccharides derived from 23F, respectively, and transport proteins, e.g. , polysaccharides 13. the CRM197 protein is a bond species-13 containing the protein conjugate pneumococcal vaccine compositions;
[32]
(2) Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and of 14 capsular polysaccharides derived from 23F, respectively, and transport proteins , for example, polysaccharides of 14 the CRM197 protein is a junction 14 comprising a protein conjugate pneumococcal vaccine compositions;
[33]
3, serotype 1, cliff Streptococcus pneumoniae 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and with the 15 kinds of capsular polysaccharide, respectively derived from 12F transport proteins, for example, CRM197 protein is a polysaccharide of the bonded 15 species-15 is Streptococcus pneumoniae vaccine composition comprising a protein conjugate;
[34]
4, Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B and the 15 kinds of capsular polysaccharide, respectively derived from transport proteins, for example, CRM197 protein is a polysaccharide of the bonded 15 species-15 is Streptococcus pneumoniae vaccine composition comprising a protein conjugate; or
[35]
(5) Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 17 kinds of capsular of derived from 33F each polysaccharide and transport proteins, for example, CRM197 protein is a polysaccharide of the bonded 17 species-17 is a protein conjugate of Streptococcus pneumoniae, including vaccine compositions
[36]
One can.
[37]
Multivalent pneumococcal vaccine compositions provided herein, for example, is the 13 of the aforementioned composition to 24, 13 is to 19, the 13 a to 17, and 13 is to 15 the, 14 a to 24, 14 have to 19 a, 14 a to 17, or 14 is to 15 the pneumococcal vaccine has a markedly superior activity compared with the polyvalent vaccine (e. g. 13 the vaccine) developed in the existing, pneumococcal infection prophylactic and / or We can expect a very good effect on the treatment.
[38]
The Streptococcus pneumoniae vaccine composition may be formulated into multidose amount (multiple dosages) for the one-time dose or multidose for one-time administration of the composition. Standing the right used herein "multidose amount" is first administered (inoculated) more than once on the object (e.g., twice or more) the administration (immunization) vaccines dose or two or more administration (immunization) the object at a time or one administered more than once (e.g., twice or greater) (inoculum) may refer to a preparation unit, including a possible vaccine dose.
[39]
The pneumococcal vaccine compositions, of two or more pneumococcal capsular polysaccharides of serotypes aureus - in addition to the protein conjugate, and a preservative. Preservatives usable in the vaccine composition (e.g., injection formulation) is 2-phenoxyethanol, formaldehyde, chlorobutanol, m- cresol, methyl paraben, propyl paraben, benzethonium chloride benzamide to, benzalkonium chloride, benzoic acid, benzyl alcohol , phenol, chimerosal, it may be at least one or more than one member selected from the group consisting of phenyl mercury nitrate, and the like.
[40]
In one example, wherein the preservative is a is the aforementioned multivalent pneumococcal vaccine compositions, for example, the previously described 13 a to 24, and 13 are to 19 and 13 are to 17 and 13 are to 15 and 14 are to 24, as a 14 a to 19 a, 14 a to 17 a, or 14 to 15 is optimized for the Streptococcus pneumoniae vaccine composition form, may be one containing 2-phenoxyethanol and formaldehyde. In this way, by including 2-phenoxyethanol with formaldehyde as a preservative, by reducing the amount of 2-phenoxyethanol toxicity and / or while still reducing the side effects, achieve more improved anticorrosion effect by the synergistic action of the two components can do.
[41]
Multivalent pneumococcal the content of 2-phenoxy ethanol included in the vaccine composition is less than 10mg / ml of the present invention, 7mg / ml, less than 6mg / ml or less, or 5mg / ml or less, for example 4mg / ml at least 10mg / ml or less, 4mg / ml to about 7mg / ml, 4mg / ml at least 7mg / ml, less than 4 to 6mg / ml, 4.5mg / ml to about 6mg / ml, 5mg / ml to about 6mg / ml, from 4 to 5mg / ml, or 4.5mg / ml and may be a 5mg / ml (used for the right stand numerical ranges as used herein, "to" is the lower limit value or more indicates the numerical range of the upper limit value or less. hereinafter the same). Content of 2-phenoxyethanol vaccine composition, it is recommended not less than the lower limit of the range, toxicity and / or side effects may be less than the upper limit value or less than the above range in order to ensure that an acceptable level to indicate the available preservative effect .
[42]
Multivalent pneumococcal content of the formaldehyde contained in the vaccine composition of the present invention is 90 to 200 ㎍ / mL, 90 to 190 ㎍ / mL, 90 to 180 ㎍ / mL, 90 to 170 ㎍ / mL, 100 to 200 ㎍ / mL , it may be 100 to 190 ㎍ / mL, 100 to 180 ㎍ / mL, or 100 to 170 ㎍ / mL. Formic aldehyde content of the vaccine composition is preferably less than or equal to the upper limit of the above range in order to ensure that it is recommended not less than the lower limit of the range, or no toxicity and / or side effects acceptable level to indicate the effective antiseptic effect.
[43]
The vaccine composition ordinary storage conditions, e.g., 2 ℃ to 8 ℃, 20 ℃ to 25 ℃, or at a temperature of about 37 ℃ about 3 months, about 6 months, at least about 1 year, at least about 1.5 years for at least about 2 years, or at least about 2.5 years it is characterized in that the stable. Infection, such as the stability of the vaccine composition herein is a vaccine composition is equal to the original antigenic (immunogenic) maintained at a level, or, and / or maintained without the components are decomposed or eliminated, and / or bacterial / viral None can mean.
[44]
Another example provides a pneumococcal infection, or preventing or treating Streptococcus pneumoniae pharmaceutical composition for infectious diseases comprising a polyvalent pneumococcal vaccine compositions previously described. Other examples are described above multivalent pneumococcal vaccine compositions of a pharmaceutically effective amount of a pneumococcal infection, or Streptococcus pneumoniae preventing or a Streptococcus pneumoniae comprising the step of administering to a subject in need infection or pneumonia the treatment of infectious diseases aureus prevention of infectious disease or provide treatment. Pneumococcal vaccine compositions used in the pneumococcus vaccine composition or the prevention and treatment included in the pharmaceutical composition may be in multidose amount of a pharmaceutical composition for a single dose pharmaceutical composition for administration of a single dose or multidose.
[45]
The pneumococcal infections is to mean any disease caused by Streptococcus pneumoniae infection, and the like pneumonia, otitis media, sinusitis, bacteremia. "Pneumonia" is a kind of acute infectious inflammation of the lung is usually Streptococcus pneumoniae ( Streptococcus pneumoniae ) and Silas keurep pneumoniae ( Klebsiella pneumoniae is). Particularly pneumococcal pneumonia is a disease that can cause all accounted for approximately 50% of pneumonia and appears severe chills, fever, cough and chest pain, sputum is often a hyeoldam effusion as a complication, meningitis, endocarditis, peritonitis, etc. ( ... Diagn Microbiol Infect Dis, 2001, 39:. 181-185).
[46]
The term "Streptococcus pneumoniae (pneumococcus)" in the present invention Streptococcus pneumoniae ( Streptococcus pneumoniae is a convenient symbiotic (commensal) organism colonization of mucosal surfaces in general, human nasopharynx (nasopharynx) refers to). When factor (factor) of the host is allowed access to the even of an organism (lower respiratory tract), occurs vigorous inflammatory response followed, thereby to cause curing (consolidation) dense when the alveolar space to fill the exudate (exudate), pneumonia the can cause. The pneumococcus can be synthesized in a unique capsular polysaccharides (capsular polysaccharide) with more than 90 structurally, this serotype (serotype) of Streptococcus pneumoniae are classified according to the structural and immunological properties of such capsular polysaccharides. Thus, when producing the back-credit formulation using a capsular polysaccharide of Streptococcus pneumoniae can appear different from the immune response according to the capsular polysaccharide type, i.e. capsular serotypes of Streptococcus pneumoniae polysaccharide is derived.
[47]
The capsular polysaccharide has, so are recognized as an antigen when administered in vivo to produce the antibody of this, it is possible to use them to produce a vaccine composition for the prevention of Streptococcus pneumoniae (S. pneumoniae infection or disease). If the term "antigen" means a substance enters the body in the present invention refers to a material that can cause an immune response specifically. One Streptococcus pneumoniae in the embodiments ( Streptococcus pneumoniae ) serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C , 19A, may 19F, 20, 22F, 23F, and 13 kinds to capsular polysaccharides derived from 24 species of 33F may function as an antigen, respectively.
[48]
Another example provides stability and / or preservative (or room buoyancy), stability of the enhanced method of producing a vaccine composition against Streptococcus pneumoniae, or Streptococcus pneumoniae vaccine, and / or preservative (or room buoyancy) enhancement method. The method
[49]
(1) Streptococcus pneumoniae ( Streptococcus pneumoniae preparing a protein conjugate, and - capsular polysaccharides (capsular polysaccharide) of a)
[50]
(2) the capsular polysaccharides comprising the steps of mixing a protein conjugate, and a 2-phenoxyethanol (2-Phenoxyethanol, 2-PE), and formaldehyde (Formaldehyde, HCHO)
[51]
It may contain.
[52]
The amount of the capsular polysaccharide, protein, conjugate, and 2-phenoxyethanol with formaldehyde are as previously described.
[53]
In the method, the capsular polysaccharides may be prepared by standard techniques known to those skilled in the art, and are not particularly limited to that method. The capsular polysaccharides may be reduced in size through a hydrolysis in order to reduce the viscosity and induces an effective immunogenic.
[54]
(1) capsular polysaccharides comprising the steps of preparing a protein conjugate may comprise the step of connecting via a carbonylation cyano (cyanylation) the capsular polysaccharide and the protein to perform a method -OC (NH) -NH-.
[55]
In one embodiment, the above-mentioned (1) capsular polysaccharides comprising the steps of preparing a protein conjugate,
[56]
(i) Streptococcus pneumoniae ( Streptococcus pneumoniae separating or isolated and purified capsular polysaccharide from a);
[57]
(Ii) decomposing dissolution and / or hydrolysis of the separated capsular polysaccharides; And
[58]
(Iii) a cyano biotinylated (cyanylation) step of the capsular polysaccharide and the protein by performing the method connected via a -OC (NH) -NH-
[59]
It may contain.
[60]
In another example, the above-mentioned (1) capsular polysaccharides comprising the steps of preparing a protein conjugate, the method comprising the capsular polysaccharide and the protein by performing the above-mentioned (iii) a cyano biotinylated (cyanylation) method connected via a -OC (NH) -NH- after this, it is possible to perform an additional ultrafiltration step, sterilizing filtration step, and at least one step selected from the adsorption step. The cyano annealing method can be performed using the CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or CNBr.
[61]
In the specific embodiment of the present invention were dissolved respectively using sodium deoxycholate (sodium deoxycholate) of Streptococcus pneumoniae having a different serotype 13 kinds or 15 kinds, was a glass the polysaccharide binding to the cells. Then 21 serotypes 1, 2, 3, 4, 5, 6A, 6B, 9V, 8, 9N, 10A, 11A, 12F, 15B, 17F, 18C, 19A, 19F, 20, 22F, and 23F-derived polysaccharide cases were possible with ionic bonding (cetyltrimethylammonium bromide) CTAB, so purification by following the CTAB step, CTAB and that does not react three serotypes 7F, 14, and 33F were purified by using the aluminum phosphate gel (Algel) solution. Reaction with the CTAB is, for example, 1 to 20% (w / v) or 5 to 15% (w / v) 0.5 to 5% by weight of the CTAB of the concentration in the reaction or from 1 to the amount of 3% by weight (e.g., for 23F polysaccharide derived from 2 to 3% by weight, in the case of the polysaccharides derived from serotypes except 23F, but may be carried out by addition of 1 to 2% by weight), without being limited thereto. After the reaction of the CTAB, after centrifugation the pellet recovered, sodium chloride solution is carried out by adding any one or more of the removal step of CTAB ion with the re-suspended, and sodium iodide in the pellets with (e. G., About 100 to 500mM) It is, but is not limited to this. The aluminum phosphate-gel reaction can be carried out by adding to the amount of 1 to 20% by weight or 5 to 15% by weight of the aluminum phosphate gel solution to the reaction, but is not limited thereto.
[62]
So if using only the capsular polysaccharides used as a vaccine composition, the immune system infants and children less than adults will not recognize it as an antigen can be an immune response will not occur, the present invention in which the conjugate form combines transport protein and the capsular polysaccharide in It was used to prepare.
[63]
The "transfer protein (carrier protein)" is herein to mean a protein which can be conjugated to capsular polysaccharides and covalently astute increase the immunogenicity of polysaccharide antigens, particularly those types are described above. CRM197 can be used in one embodiment. The transport proteins can be joined to the capsular polysaccharide through a standard bonding method, the capsular polysaccharide is formed through which-carrying protein conjugate may be one or more capsular polysaccharide is conjugated to a transport protein.
[64]
Capsular polysaccharide and a known method for producing a joined body of the transport protein is all may be included in the scope of the present invention, the conjugate is a cyano carbonylation method capsular polysaccharide and protein transport using the associated group -OC (NH) -NH- It has a structure. The cyano carbonylation method using methods known to those skilled in the art, and can be suitably carried out, for example, but is not CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or, but may be performed using a CNBr, limited.
[65]
Capsular for example for the production of conjugates of polysaccharides and protein transport, may form a conjugate (glycoconjugate) sugar by chemical activation with the purified capsular polysaccharides and, one joined to each capsular polysaccharide is chemically activated to transport proteins. CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) cyano activity by a carbonyl illustration by processing and ah change carbonate group between the hydroxy groups of the capsular polysaccharide, by using this, to form the amino group with a covalent bond of the carrier protein CRM197 can. Cyano carbonyl migration induced by the CDAP is not necessarily specifically by the addition of glycine (glycine) solution of 3 molar equivalents compared CDAP 1 molar equivalent, and can be to terminated by adjusting the pH to 9.0, however, limited to those skilled in the art that purpose It can be appropriately regulated in accordance with the reaction solution and the reaction conditions.
[66]
The resulting capsular polysaccharide-protein conjugate is carried can be purified by a variety of methods. Examples of these methods include concentration / diafiltration process, a column chromatography and a multi-layer filtering. The purified polysaccharide-protein conjugate can be formulated into vaccine compositions of the present invention by mixing, respectively, and use them. It may perform the formulation of a vaccine composition of the present invention using the recognition method in the art. For example, the individual capsular polysaccharides of 13 species can be produced by a composition formulated with an acceptable vehicle carrying protein conjugate physiologically. Examples of such vehicles include water, buffered saline, water for injection, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol), or may be a dextrose solution, without being limited thereto.
[67]
In the specific embodiment of the present invention, 1) dissolution and hydrolysis of capsular polysaccharide 13 kinds of degradation, 2) CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) each joining reaction step of the capsular polysaccharide and CRM197 using 3) joining the end of reaction, 4) ultrafiltration and 5) sterilizing filtration, and 6) capsular polysaccharide through the adsorption step - the transport protein conjugates were prepared 13 kinds.
[68]
The term "vaccine" in the present invention is a biological preparation containing an antigen to the immune to a living body, it means the immunogenic or antigenic substance that causes immune to the living body by administering to humans or animals for the prevention of infectious diseases.
[69]
The vaccine composition, is aejyu adjuvant, preservatives, buffering agents, freezing agents, salt, 2 to be further comprises at least one selected from the group consisting of cationic, non-ionic detergent and a free radical oxidation inhibitor.
[70]
The term "adjuvant" herein refers to substances used to increase the immunogenicity of the immunogenic compositions of the present invention. The adjuvants are often provided in order to promote an immune response, which is well known to those skilled in the art. Suitable adjuvants to enhance the effectiveness of the vaccine composition of the invention, but may include at least one selected from the group consisting of, without being limited thereto:
[71]
(1) aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate and the like);
[72]
2-water emulsion formulations (Mura wheat peptide (not containing other specific immunostimulants such as defined below) or bacterial three pobyeok components or content), for example (a) MF59 (No. WO90 / 14837 No.) : 5% (w / v) squalene (squalene), 0.5% (w / v) tween (tween) 80 and 0.5% (w / v) span (span) containing 85 and (optionally various amounts of MTP-PE may further contain), Model 110Y micro fluidizer low (microfluidizer, Microfluidics, Newton, MA) the micro fluidizer formulations using low to sub-micron particles hwadoem, such as (b) SAF: 10% (w / v ) squalene, 0.4% (w / v) tween 80, 5% (w / v) Pluronic (pluronic) - and containing a block polymer L121, and refer to thr-MDP (below), micro-fluidization in the submicron emulsion ( microfluidization) or, Sikkim to form a larger particle size emulsion of the cavity, and (c) Libby (Ribi) ™ adjuvant system (RAS) (Corixa, Hamilton, MT): 2% (w / v)'s Allen, 0.2% (w / v) Tween 80 and, U.S. Patent No. 4,912,094 a technique for 3-O- talah No. acylated mono phosphoryl lipid A (MPL ™) (Corixa), trehalose dimi cholate (TDM), and cell wall skeleton one or more bacterial cell wall components from the group consisting of (CWS), preferably also containing MPL + CWS (Detox (Detox) ™);
[73]
3, Quill A (Quil A) or styryl myulron (STIMULON) jjye QS-21 (Antigenics, Framingham, MA, United States Patent No. 5.05754 million call) and the saponin adjuvant is used or such a particle generated therefrom (for example: ISCOM ( immunostimulatory complex));
[74]
(4) bacterial lipopolysaccharide, synthetic lipid A analogs (such as aminoalkyl glucosamine phosphate compounds (AGP)), or may be obtained from derivatives or analogs (which its Corixa, U.S. patent that the described in the 6,113,918 call; the AGP An example of the 2 - [(R) -3- tetra decanoyl oxy tetra decanoyl amino] ethyl 2-deoxy -4-O- phosphono -3-O- [(R) -3- tetra decanoyl oxy tetra decanoyl] -2 - [(R) -3- tetra decanoyl oxy tetra decanoyl amino] and -bD- glucopyranoside, which is also known as 529, and (also known previously to as RC529), which can be molded or formulation hwadoem a stable emulsion);
[75]
(5) synthetic polynucleotides (such as oligonucleotides containing a CpG motif nucleotides (U.S. Patent No. 6,207,646));
[76]
(6) cytokines, e.g., interleukins (such as: 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 .;
[77]
7 wild-type cholera toxin (CT) or, for example, in WO-2000/18434 call is glutamic acid in the amino acid position 29 other amino acids in accordance with, specifically substituted with histidine, a mutant type cholera toxin (the WO -2002/098368) and (WO-2002/098369 No.), pertussis toxin (PT), or Escherichia coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PTK9 / G129 (the WO -93/13302 call and a detoxified mutant of a bacterial ADP- ribonucleic misfire toxin, such as the WO-92/19265 Lake); And
[78]
8, such as a trimer of complement component C3d Complement.
[79]
The unevenness wheat peptide is N- Acetyl-mura wheat -L- tray O'Neill -D- iso glutamine (thr-MDP), N- acetyl-NORD-mura wheat -L- alanine-2- (1'-2 'di-palmitoyl a -sn- glycero-3-hydroxy-phosphoryl-oxy) -ethylamine (MTP-PE), etc. may be included, but is not limited thereto.
[80]
The aluminum salt adjuvant is an aluminum-adsorbed vaccine may be a (alum-adsorbed vaccine) - precipitated vaccine (vaccine alumprecipitated) or aluminum. Aluminum salt is hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Al-hydrogel, Superfos, cancer pojel, aluminum hydroxide, aluminum hydroxy phosphate sulfate (Aluminum Phosphate Adjuvant (APA)), It may be included, such as amorphous alumina, without being limited thereto. APA refers to a suspension of aluminum hydroxy phosphate. Aluminum chloride and the sodium phosphate 1: After mixing at a ratio of 1: 1 (by volume) of aluminum hydroxy phosphate and sulphate is precipitated, using the High shear mixer the precipitate size, and dialyzed and then into the saline solution such that 2 ~ 8 ㎛ It may be prepared by sterilization. In one embodiment, using a commercially available Al (OH) 3 (for example, Al hydrogel or Superfos) to adsorb the protein. The protein of 50 ~ 200 g per 1 mg of aluminum hydroxide can be adsorbed, and the ratio is dependent on the pH of the pI of the protein and the solvent. Low pI protein is strongly bonded to the protein, compared with a high pI. Aluminum salts can form an antigen depot 2-3 weeks slowly release the antigen by macrophages into non-specific, activation of complement, the innate immune mechanisms.
[81]
The term "preservative (or preservatives)" in the present invention, wherein for suppressing the growth of microorganisms in the vaccine composition refers to a material that the virus and / or antibacterial action and, for example chimerosal (thimerosal), phenoxyethanol ( 2-phenoxyethanol), formaldehyde (formaldehyde), or all the usual preservatives a mixture thereof, but is used in the art without limitation can be used.
[82]
In addition, the vaccine composition may comprise one or more buffering agents acceptable, physiologically. For example, if the vaccine composition of claim injection (infusion) or the first scanning buffer may be one having a pH 4.0 to 10.0, particularly, 5.0 to 9.0 pH, buffering capacity at pH 6.0 to 8.0 in more detail. The buffer may be at least one selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tart rate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, triethanol amine buffer .
[83]
In particular, to the purpose of administering the vaccine composition of the invention parenterally, the buffer may be selected from buffering agents suitable for USP. For example, buffering agents include acetic acid, benzoic acid, gluconic acid, glyceric acid, lactic acid and a monobasic acid such as; Dibasic acids such as aconitic acid, adipic acid (adipic), ascorbic acid, carbonic acid (carbonic), glutamic acid, malic acid, succinic acid, tartaric acid; Polybasic acids such as citric acid, phosphoric acid; One member selected from the group consisting of a base such as ammonia, diethanolamine, glycine, triethanolamine, TRIS may be equal to or greater than.
[84]
In addition, the vaccine composition of the present invention may include a non-ionic detergent. For example, polyoxyethylene sorbitan esters (usually called the Tweens), especially polysorbate 20 and of polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO) (example: DOWFAXTM); Ethoxy (oxy-1,2-ethanediyl) the number of repeating ethoxy groups different Oaks nolryu, especially agarose ethoxy play -9 (Triton-100); Ethyl phenoxy polyethoxy ethanol (IGEPAL CA-630 / NP-40); Phospholipids such as lecithin; Ethoxylated nonylphenol, such as NP series; Lauryl, cetyl, stearyl, oleyl a polyoxyethylene fatty acid derived from an alcohol ether (Brij surfactants), in particular triethylene glycol mono-lauryl ether (Brij 30); It can include a surface active agent such as sorbitan ethers, especially sorbitan trioleate (Span 85) and sorbitan monolaurate, known as the SPANs However, without being limited thereto. Tween 80 may also be used a mixture of non-ionic detergents such as the number and, Tween 80 / Span 85 included in the emulsion. Polyoxyethylene combination of loam resorcinol such as sorbitan ester and Triton X-100 is also suitable, it is also useful combinations of Laureth 9 and Tween, and loam or resorcinol, such as Tween 80. Specifically, polyoxyethylene sorbitan esters (such as: Tween 80) to 0.01% (w / v) to 1% (w / v), especially 0.1% (w / v); Octyl phenoxy polyoxyethylene ethanol, or nonylphenoxy polyoxyethylene ethanol (example: Triton X-100) is 0.001% (w / v) to 0.1% (w / v), in particular zero. 005% (w / v) to 0.02% (w / v); Polyoxyethylene ethers (such as: laureth 9) is 0.1% (w / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v), especially 0.1% (w / v) to (w / 1% v) or may include from about 0.5% (w / v).
[85]
The vaccine composition of the invention may be formulated in a single dose vial, multidose vials or pre-filled syringe capacity (prefilled syringe) form. Thus, in one example provides a vial or a prefilled syringe containing the above-mentioned vaccine compositions in single dose or multidose, for example, greater than dose once. The vaccine composition may be further comprising a physiologically acceptable carrier.
[86]
Standing the right used herein, "multidose capacity" or "multidose amount" is first administered (inoculated) individual to once exceed administration (immunization) vaccines capacity or 2 administered over (inoculation) on the object once or 1 more than once administered (vaccination) may be available, which means the vaccine dose.
[87]
A carrier physiologically acceptable for use in liquid formulations, there are aqueous or non-aqueous solvents, suspensions, emulsions, oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, ethyl oleate. The aqueous carrier may include water, alcohol / aqueous solvent, emulsions or suspensions, saline, buffer solution. Examples of oils are lipids derived from synthetic oils, milk, egg plants, such as gender or animal oils, peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, fish keeping (marine oil). The vaccine composition of the invention is isotonic, hypertonic, or may holy days storage, the pharmaceutical composition is administered by infusion or injection is not to be essentially isotonic is preferably limited to one. On the other hand, there is isotonic or hypertonic be advantageous for the storage of the composition. If the holy the vaccine composition failure can be diluted to isotonic prior to administration. Isotonic agents for the dilution may be a ionic isotonic agent or a non-ionic isotonic agent such as a carbohydrate, such as a salt. Ionic isotonic agents include, but sodium chloride, calcium chloride, potassium chloride, magnesium chloride, etc., without being limited thereto. A non-ionic isotonic agents include, but not intended to include sorbitol, glycerol, limited.
[88]
The amount of the conjugate in each vaccine dose, may be selected as an amount which induces an immunoprotective response without significant side effects, such amount may vary depending upon the serotype of Streptococcus pneumoniae. Specifically, the vaccine composition is compared to capsular polysaccharide by weight of the serotypes 1, derived from (i. E., Relative to 1 part by weight), serotype 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A , 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, if the resulting capsular polysaccharide, respectively 0.8 to 1.2 or 0.9 to 1.1 weight ratio (i.e., 0.8 to 1.2 wt% or 0.9 to 1.1 of parts by weight) If the polysaccharide containing and serotype 6B derived capsular as is 1.6 to 2.4, 1.8 to 2.2, or a weight ratio of 1.9 to 2.1 (i.e., 1.6 to 2.4 parts by weight, 1.8 to 2.2 parts by weight, or from 1.9 to 2.1 wt. but may include a unit), it is not limited.
[89]
One example for the production of conjugates, each conjugate in the range of 0.1 to 100 ㎍, particularly from 0.1 to 10 ㎍, more specifically, may include a polysaccharide from 1 to 5 ㎍.
[90]
Further, and most specifically from other than the capsular polysaccharides of serotypes 6B derived remaining polysaccharides, i.e. serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B , 17F, 18C, 19A, 19F, 20, 22F, 23F, and capsular polysaccharides 33F derived respectively from 2 to 2.4 ㎍ or 2.1 to 2.3 ㎍, for example, in an amount of from about 2.2 ㎍, capsular polysaccharide of serotype 6B derived from 4 to 4.8㎍, 4.2 to 4.6 ㎍, or 4.3 to 4.5 ㎍, for example, may be included in an amount from about 4.4 ㎍, but is not limited thereto. In this case, in the composition CRM197 protein is in the range of 0.1 to 100 ㎍, 1 to 50 ㎍, 20 to 40 ㎍, or 28 to 31 ㎍, for example, may be included in an amount from about 29.3 ㎍, but is not limited thereto.
[91]
The optimum amount of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. For example, you can determine the capacity of a vaccine to human subjects by extrapolating the results of animal experiments. In addition, those skilled in the art Empirically, one can determine the dose, if necessary.
[92]
The vaccine composition may be further comprising an aluminum element and a sodium chloride, but not limited to this.
[93]
A vaccine composition according to the present invention can be used by administering a pharmaceutically effective amount of a systemic or mucosal route, protection for easy object susceptible to pneumococci and prevent pneumococcal disease. The term "prevention" of the present invention by administration of the vaccine composition of the present invention refers to all activities that inhibit or delay the infection by the pneumococcus. To induce a "pharmaceutically effective amount of a" is an antibody of can significantly reduce the severity of the infection probability or degree of infection in Streptococcus pneumoniae, which is defined in the present invention refers to the dose required. The term "administration" of the present invention refers to the introduction of a predetermined material on an object by any suitable method. The vaccine composition of the present invention should not be oral, nasal, rectal, may be administered to the suction path through the transdermal or aerosol, the dosage or as a bolus, but may slowly main import, limited. The administration is intramuscular, intraperitoneal, intradermal or subcutaneous injection through a path; Or it may be selected from the mucosa of the mouth / digestive tract, airway tube or genitourinary tube. In one embodiment, it is possible that the intranasal be used for the treatment of pneumonia or otitis media, in this case, to more effectively prevent the nasopharynx colonization of S. pneumoniae, it is possible to weaken the infection at an early stage.
[94]
Administration subject a vaccine composition of the invention or a pharmaceutical composition, "objects" may be one which means a cell, tissue or culture isolated from a living organism or which containing pathogens can infect the organism can be a higher vertebrates number and, more specifically, but can be a mammal such as a human, it is not particularly limited.
[95]
In another embodiment, the compositions of this invention may be administered in a single vaccination, but twice at appropriate intervals, can be administered three times, four times or more, but is not limited thereto. For example, a routine immunization in infants and newborn child for invasive disease caused by Streptococcus pneumoniae plan may be the age of 2, 4, 6, and 12 to 15 months.
[96]
In addition, the composition may further include one or more proteins derived from the cliff Streptococcus pneumoniae. Examples of suitable Streptococcus pneumoniae proteins for inclusion, as well as the protein described in the WO-2002/053761 Ho, both also a protein identified in the WO-2002/083855 Ho may be included in the scope of the invention.
[97]
In the specific embodiment of the invention, a total of 0.5 mL vaccine composition, of 2.2 ㎍ each polysaccharide, derived from stage 6B polysaccharide is 4.4 ㎍; CRM197 transport protein of about 29.3 ㎍; 0.5 mg of aluminum element (2 mg aluminum phosphate) adjuvant; Sodium chloride, about 4.25 mg (if the preservative is not included), or from about 3.5 mg (For comprises a preservative); Succinate buffer, about 295 ㎍; And 2-phenoxyethanol polyhydric containing from about 3 mg and about 60 ㎍ formaldehyde, for example, the 13 a to 24 a, 13 a to 19 a, 13 a to 17 a, 13 a to 15 a to 24 14 a, 14 a to 19, and 14 a to 17, or 14 to 15 is a pneumococcal vaccine compositions can be given.
[98]
In another specific embodiment of the present invention in the serum of the rabbit inoculated with the vaccine composition, it was confirmed the high levels of serotype-specific IgG concentrations than Prevenar 13® (Table 1). In addition, it was confirmed that an excellent effect than seen in Prevenar 13® functional immunogenic Identification (Opsonophagocytic assay) for it (Table 2). Thus, vaccine compositions of the invention can be seen that this is a very excellent effect in preventing the use of pneumococcal disease.
[99]
Another aspect of the present invention capsular polysaccharides (capsular polysaccharide) - transport protein conjugates 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 species, an immunogenic composition for Streptococcus pneumoniae, including 14 species to 17 species, or 14 species to 15 species. The conjugates and Streptococcus pneumoniae are as described above.
[100]
Capsular polysaccharide of the invention carry protein conjugates 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds to 17 kinds , or a composition comprising 14 kinds to 15 kinds are 13 kinds to 24 kinds, 13 kinds to 19 kinds, 13 kinds to 17 kinds, 13 kinds to 15 kinds, 14 kinds to 24 kinds, 14 kinds to 19 kinds, 14 kinds of to 17 species, or a Streptococcus pneumoniae having 14 kinds to 15 kinds of different serotypes of (Streptococcus pneumoniae) it comprises an origin capsular polysaccharide, to be recognized by the antigen when administered it in the body to produce antibodies for this bar causes an immune response, can be used as immunogenic compositions against S. pneumoniae.
[101]
Another aspect of this invention is a method for preventing pneumococcal disease by administering to a subject in need thereof the vaccine compositions or immunogenic compositions.
[102]
Another aspect of the present invention capsular polysaccharide-carrier comprises a protein conjugate of 13 species, wherein the conjugate of the 13 species Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, the 14, 18C, 19A, 19F and 23F will the respective 13 kinds of capsular polysaccharides (capsular polysaccharide) of the resulting bonded covalently to the transport protein, wherein the transport protein is CRM197 protein and the conjugate is cyano carbonylation method using the capsular polysaccharide and the protein is carried to provide a use for use in the preparation of vaccine compositions for one of, a composition Streptococcus pneumoniae disease prevention of having a structure connected to a group -OC (NH) -NH-.
[103]
For such vaccine compositions, immunogenic compositions, and the prevention of pneumococcal disease as described above.
[104]
Another aspect of the invention Streptococcus pneumoniae (Streptococcus pneumoniae) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and discrete capsular 13 kinds of 23F-derived polysaccharides (capsular polysaccharide) to a cyano carbonylation method, method for producing the immunogenic composition, comprising the step of capsular polysaccharide and CRM197 protein carrying the junction to have a structure connected to a group -OC (NH) -NH- using each.
[105]
For immunogenic compositions and methods for their preparation as described above.
Effects of the Invention
[106]
Multivalent pneumococcal vaccine compositions according to the present invention capsular polysaccharides of the specific bonding structure by including a 2-phenoxyethanol and formaldehyde with a multivalent conjugate of the protein, and thus optimize the combination and content as a preservative, excellent immunogenicity It characterized by an excellent stability and / or retention maintained. Thus, vaccine compositions and immunogenic compositions according to the present invention can be used more safe and useful for the prevention of disease caused by Streptococcus pneumoniae of zero, infants, children, and adults.
Mode for the Invention
[107]
Hereinafter will be described the present invention in more detail by the following examples. However, these are only intended to illustrate the invention, but is not the scope of the present invention is limited by these examples.
[108]
Preparation Example 1: Preparation capsular polysaccharide
[109]
1-1. Preparation of cell banks
[110]
Depositary the Streptococcus pneumoniae with the 16 kinds of different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B) firm were obtained from the US CDC (Center for Disease Control and Prevention, US), the cell bank was prepared in the same way as you.
[111]
To smear the Streptococcus pneumoniae strains on blood agar plate confirmed the pneumococcal and thereby remove the old medium components. After selection of single colonies growing good for more than 10 single colonies were inoculated and cultured in a liquid medium (Soytone (Kerry Bio-Science), or Yeast extract (Bio-springer) derived medium) that does not contain components of animal origin, proliferation after that, was prepared by adding a synthetic glycerol, a research cell bank containing synthetic glycerol (research cell bank, RCB).
[112]
Of the polysaccharide expression having individual serotypes identified research cell bank was taken out one vial after proliferation the cells in a liquid medium that does not contain components of animal origin by adding a synthetic glycerol was prepared in a master cell bank, a master cell taken out of bank one vial was prepared for producing cell banks and after proliferation the cells in a liquid medium that does not contain components of animal origin adding synthetic glycerol.
[113]
Preparative a cell bank was used for the test to be kept in a frozen state in seconds, of less than -70 ℃.
[114]
1-2. Polysaccharide fermentation and separation
[115]
By preparative cell by thawing one vial of the bank it was inoculated on the liquid medium that does not contain components of animal origin. Until reaching a predetermined cell density (Optical Density, OD 600) it was subjected to seed culture at 37 ± 2 ℃ to Shamanism half state. After confirming the completion of the pollution if the seed culture was completed the culture was inoculated in a fermentor containing a liquid medium (Soytone (Kerry Bio-Science), or Yeast extract (Bio-springer) derived medium) that does not contain components of animal origin (OD600 = 2.5 ± 0.2).
[116]
Then, at 37 ± 2 ℃ using a potassium hydroxide solution was sterilized with a minimum of agitation, while maintaining the pH of the medium to 7.2 ± 0.2 was subjected to main culture. Samples after initiation of the culture 2 hours to measure the concentration of glucose contained in the culture medium and the cell density in the culture medium. On the start of the glucose in the medium it was depleted terminate the incubation.
[117]
After the culture was completed, and then prepared to a sterile 12% (w / v) sodium deoxycholate (sodium deoxycholate) in an appropriate amount so as to have a final concentration of 0.12% (w / v) was dissolved in the cell is added to the culture of cells the polysaccharide was coupled to glass.
[118]
1-3. Purification of capsular polysaccharides
[119]
After the above-mentioned Production Examples 1 to 3, sodium deoxycholate, appropriate after 15 hours the pH of phosphoric acid in the treated sample with 85% phosphoric acid was added after a long period of time by 3.5 ± 0.3 ± 3 sigan value in response obtained during centrifugation (17,000 through xG, room temperature, 1 hour) to recover the supernatant. The recovered supernatant Depth filter (depth filter) - passed to (0.55 9.0 um) and then, concentrated, and proceeding to the buffer exchange to PBS.
[120]
After buffer exchange was conducted in which the following, removal of impurities in two ways such as to pass through the sample to a carbon carbon filter (active carbon filter):
[121]
Since 1) serotype 1, 2, 3, 4, 5, 6A, 6B, 9V, 12F, 15B, 18C, 19A, is possible if the 14 serotypes CTAB (cetyltrimethylammonium bromide) and ionic bonding of 19F and 23F, was in progress the CTAB step was specifically 10 weight% CTAB solution to 1.5% by weight of the process solution weight ratio (in the case of non-23F) or 2.5% (in case of 23F) 1 sigan ± 10 min treatment in an amount of reaction. After centrifugation for one hour at a rate of 17,000 xG Pellet it was collected. Then, sodium chloride (NaCl) and using the 200 ~ 300mM sodium chloride (NaCl) solution to a sodium iodide (NaI) processes haejugo Resuspension a Pellet number of times through the CTAB treatment, iodide equivalent to 50% by weight of CTAB amount with sodium (NaI) was removed CTAB ion. However, in the case of serotype 3 it was used 350mM NaCl solution. After the sodium chloride and sodium iodide treatment to recover the supernatant from the centrifugation was used in the next step. 2) In the case of the two serotypes of the serotypes 7F and 14 that do not react with CTAB, a reaction by the addition of aluminum phosphate gel (Algel) solution was then treated with aluminum phosphate gel solution containing 10 wt% of the total process solution over 1 time and reacted. To recover the supernatant obtained by centrifugation (1 ㅣgan centrifugation at 17,000 xG conditions) was used in the next step.
[122]
The two types of samples completing the impurity removing process is subjected to Depth filter (Depth filter) and ultrafiltration (UF / DF) step, and then, controls the amount of ethanol and sodium chloride, and was stored as a powder concentrate to create the form.
[123]
Preparation Example 2: Streptococcus pneumoniae capsular polysaccharide-protein conjugate prepared
[124]
2-1: Comparison laminating method of reductive amino-speech and speech cyano carbonyl
[125]
Purification of serotype 9V polysaccharide and CRM197 carrier protein (GenBank Accession No. 1007216A; SEQ ID NO: 1; mgaddvvdssk sfvmenfssy hgtkpgyvds iqkgiqkpks gtqgnydddw kefystdnky daagysvdne nplsgkaggv vkvtypgltk vlalkvdnae tikkelglsl teplmeqvgt eefikrfgdg asrvvlslpf aegsssveyi nnweqakals veleinfetr gkrgqdamye ymaqacagnr vrrsvgssls cinldwdvir dktktkiesl kehgpiknkm sespnktvse ekakqyleef hqtalehpel selktvtgtn pvfaganyaa wavnvaqvid setadnlekt taalsilpgi gsvmgiadga vhhnteeiva qsialsslmv aqaiplvgel vdigfaaynf vesiinlfqv vhnsynrpay spghktqpfl hdgyavswnt vedsiirtgf qgesghdiki taentplpia gvllptipgk ldvnkskthi svngrkirmr craidgdvtf crpkspvyvg ngvhanlhva fhrsssekih sneissdsig vlgyqktvdh using tkvnsklslf feiks), attempts to laminating method of reductive amino speech (reductive amination), and cyano carbonyl speech (cyanylation) to compare the bonding yield of the two bonding methods. Specific bonding process is as follows.
[126]
2-1-1. Reductive amino speech
[127]
By putting 11.7mg of periodic acid salt (Sodium periodate) to the stock solution of the polysaccharide serotypes 9V with stirring at 21 to 25 ℃ it was activated polysaccharide. After the oxidized polysaccharide was concentrated and diafiltration using an ultrafiltration filter with WFI (water for injection) of 100 KDa, mix the remaining residual polysaccharide to CRM197 protein and the saccharide /CRM197=0.5 (by weight) ratio and lyophilized. Thawing the freeze-dried complex and was stabilized (equilibrated) at 21 to 25 ℃. The equilibrated composites of sodium phosphate (Na in a ratio of saccharide 0.1M per 20g 3 PO 4 was dissolved by incubation (37 ± 2 ℃) in a) buffer solution, by a cyanoborohydride In the hydride (100mg / mL) and protein It was initiated the bonding reaction between the sugar. On the 37 ± 2 ℃ incubated for about 44 to 52 hours, cooled to 23 ± 2 ℃ to 0.9% (w / v) NaCl solution 1mL was added to the reactor. Sugars 1 to the sodium beam so that the hydride of 1.8 to 2.2 molar equivalents of sodium beam per mole of hydride solution was added (100mg / mL) and stirred the reaction mixture obtained at 23 ± 2 ℃ and the incubation, present in the saccharide It was reduced any unreacted aldehydes. The obtained saccharide-bonded protein mixture 0.9% (w / v) was diluted by the addition of aqueous sodium chloride solution in 5mL and, the mixture was diluted bonded dialysis and filtration using a 100 kDa MWCO membrane.
[128]
2-1-2. Cyano carbonyl speech
[129]
By the addition of sodium chloride stock solution of the polysaccharide powder for serotype 9V prepared without hydrolysis process to prepare a 2M NaCl polysaccharide solution. In order to activate the polysaccharide, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) solution was added to serotype 9V polysaccharide solution to a 0.5% (w / w) concentration compared to the polysaccharide, the polysaccharide activation reaction was stirred for 15 minutes It was induced. To then rise until the pH is 9.5 ± 0.1 ℃ to the sodium hydroxide solution was added to the obtained reaction mixture, the hydroxyl groups of the polysaccharide to be fully activated by CDAP was stirred for 3 minutes. 1.0% of the CRM197 polysaccharide activation process in a rough proportion of polysaccharide compared to the polysaccharide solution CRM197 (w / w) (CRM197 weight / weight polysaccharide) was allowed to proceed the bonding reaction at room temperature for 1 hour by the addition so that the.
[130]
The conjugation reaction is 2M glycine (glycine) solution was added to 3 molar equivalents compared CDAP 1 molar equivalent, and the pH was adjusted to 9.0 to terminate the reaction by incubation overnight at room temperature. Completion of the reaction, the conjugate is then concentrated and diafiltration on an ultrafiltration filter with a buffer solution including 0.9% (w / w) sodium chloride.
[131]
As a result, produced by the carbonylation method cyano conjugate it was confirmed that the yield is more than 4 times that of the conjugates prepared by reductive amination method. Thus, the present inventors have prepared a conjugate using capsular polysaccharide and CRM197 by cyano carbonylation method.
[132]
2-2. Purification of the conjugate bond and
[133]
Streptococcus pneumoniae capsular conjugate Preparation of Paracoccus polysaccharide and CRM197 were carried out via the process of the following steps:
[134]
Step 1. decomposed and dissolved singer of capsular polysaccharides
[135]
A capsular polysaccharide powder concentrate derived from each of the serotypes have a final concentration range was filtered through a 0.45 ㎛ Each filter was dissolved, and a water for injection so that the range described below:
[136]
1) In the case of serotype 1, 2 and 4, 0.8 to 2.0 mg / ㎖ range,
[137]
2) the range of serotypes 5, 6B, when the 9V, 18C and 19F, 4 to 8 mg / ㎖,
[138]
3) The range of serotypes 6A, 12F and 19A is 8 to 12 mg / ㎖, and
[139]
4) range from serotypes 3, 7F, 14, 23F and 15B of the case, 2 to 4 mg / ㎖.
[140]
The solution in the pH and temperature ranges set forth below for each serotype was carried out the step of incubating:
[141]
1) serotype 1, 2, 4, 5, 6B, 7F, 14, and 23F of the case, 70 to 80 ℃ overnight,
[142]
2) For serotypes 6A and 19F, 70 to 80 ℃ for 0.5 to 4 hours,
[143]
3) performing the incubation process in serotype 3, 9V, 12F, and the case of 18C, pH 2.0 and 65 to 80 ℃ for 0.5 to 4 hours by using a phosphoric acid solution.
[144]
4) In the case of serotype 15B, 19A, it does not perform hydrolysis.
[145]
That was then cooled to 21 ℃ to 24 ℃ and stopping the hydrolysis by the addition of sodium hydroxide so that the target pH of 6.0 ± 1.0.
[146]
Step 2. Joining the reaction process of the capsular polysaccharide and CRM197
[147]
It was added to sodium chloride powder to all serotypes was prepared in a 2M NaCl polysaccharide solution. The appropriate CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) in a solution of 1/1 acetonitrile / water for injection (v / v) ratio of 100 mg per 1 ㎖ CDAP solution for each serum with the amount described below for each serotype It was added. In detail
[148]
1) In the case of serotype 6A, 9V and 14, 1 CDAP to polysaccharide compared to 1.5 (w / w),
[149]
2) In the case of serotypes 2 and 4 CDAP polysaccharide preparation 2 (w / w),
[150]
3) In the case of serotypes 1, 3, 7F, 15B, 19F, and when the 19A polysaccharide compared CDAP 3 (w / w), 4) serotypes 5, 6B, 18C, 23F CDAP contrast polysaccharides 4 (w / w) and
[151]
5) was dissolved in the case of serotype 12F polysaccharide compared CDAP 5 (w / w) ratio and added to each polysaccharide solution.
[152]
Then, the mixture was stirred for 3 to 7 minutes to then raised to pH 9.5 by addition of sodium hydroxide solution, the hydroxyl groups of the polysaccharide to be fully activated by CDAP. By the addition of CRM197 in an amount of polysaccharide compared to CRM197 0.75 (w / w) of each serotype polysaccharide solution, and proceeding the conjugation reaction for 2 hours. Thereafter, the reaction conversion rate was measured by using a SE-HPLC was added to CDAP added, if necessary.
[153]
Step 3. Joining of the reaction
[154]
, The addition of glycine (glycine) solution of 3 to 6 molar equivalents compared to the CDAP was added 1 molar equivalent for every serotype, and the reaction was terminated by adjusting the pH to 9.0. After the bonding solution was stirred one hour at 21 ℃ to 24 ℃, and stored overnight at a low temperature of 2 to 8 ℃.
[155]
Step 4. ultrafiltration
[156]
The diluted mixture using the joint (5mM Succinate buffer solution (pH 5.8) containing 150 mM NaCl) buffer, at least 20 volume was concentrated, and diafiltration on an ultrafiltration filter. Here and kept at the range of pH 5.5 to 6.5 with a buffer solution, it was used as a buffer solution including 0.9% (w / v) sodium chloride. Cut-off molecular weight of ultrafiltration the filter was performed using a 300 kDa in all serotypes, the permeate was discarded.
[157]
Step 5. sterilizing filtration
[158]
By using a (5mM Succinate buffer solution (pH 5.8) containing 150 mM NaCl) and the residue solution after diafiltration buffer solution diluted to less than 0.4g / L to polysaccharide content concentration standard it was filtered through a filter 0.22㎛. A control (saccharide content, residual DMAP) in the manufacturing process was carried out on the filtered product. To the control of the manufacturing process for the filtered residual liquid was carried out to determine whether additional concentration, diafiltration and / or dilution is required.
[159]
Step 6. Adsorption
[160]
Sikimyeo filtering filtrate aluminum salts (aluminum phosphate) to a final concentration in the adsorption was added to the 1 mg / mL of aluminum ion basis, to maintain the pH range of 5.5 to 6.5 were added to the excess salt. Adsorption stock solution is completed is subjected to quality check the quality inspection compliance, which was stored in a refrigerator for 2 to 8 ℃ until use.
[161]
Example 1. The formulation of multivalent pneumococcal conjugate vaccine
[162]
Preparative Example 2, the Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, of 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B derived from capsular polysaccharides prepared from each and the capsular polysaccharides combined with CRM197 protein - approached the following combinations from a protein conjugate pneumococcal polysaccharide-protein conjugate was prepared:
[163]
(1) 13 is bonded: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;
[164]
(2) 15-A is bonded: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 12F;
[165]
(3) 15 B is the conjugate: serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B.
[166]
The necessary amount of bulk conjugate solution was calculated based on the batch volume (batch volume) and the polysaccharide concentration of a conjugate stock solution. As in step 6 of Preparation Example 2, was added to the conjugate bulk solution of each serotype was added after the addition of aluminum phosphate, 0.85% (w / v) physiological saline solution and 5 mM succinate buffer (pH 5.8), 0.85% sodium chloride, was prepared bulk solution of each of the conjugates containing 5 mM succinate buffer (pH 5.8) and 1 mg / mL aluminum element (Al element concentration in the aluminum phosphate). Finally chimerosal as preservatives (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 to prepare a 100, 120, or 170 ug / ml) was added and the formulation was gradually mixed into the above-described combination of serotypes Chemistry multivalent pneumococcal conjugate vaccine compositions. The concentration of each serotype capsular polysaccharide of the multivalent vaccine composition is 4.4 ㎍ / mL (only Type 6B is 8.8 ㎍ / mL). When checking the pH and, if necessary, the pH was adjusted to 5.8. The final vaccine composition formulated stock solution prepared above was charged into Type1 borosilicate glass vial. The compacted vaccine composition was stored at 2 to 8 ℃.
[167]
Example 2: Evaluation of polyvalent immunogenic pneumococcal conjugate vaccine
[168]
In the embodiment in which the vaccine compositions prepared from 1, 13 is (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) vaccine, vaccine preparation Total while 0.5 mL, each capsular polysaccharide of 2.2 ㎍ (only serotype 6B capsular polysaccharide is 4.4 ㎍); CRM197 transport protein of about 29.3 ㎍; 0.5 mg of aluminum element (2 mg aluminum phosphate) adjuvant; Sodium chloride, about 4.25 mg; Succinate buffer, about 295 ㎍; The selection of the 2-phenoxyethanol from about 3 mg, and formaldehyde about 60 ㎍ a multivalent pneumococcal vaccine containing the composition (a name card LBVE013), evaluated whether having the ability to induce an immune response in rabbits. The immunogenicity is, if serum IgG levels of antigen was identified via the by specific ELISA, and antibody option Sono digging cytokine analysis system (Opsonophagocytic assay, OPA) for function.
[169]
The Prevenar 13® (Prevenar13®) used in the vaccine composition, or the positive control LBVE013 planned human clinical dose (2.2 ㎍ each polysaccharide exception: 6B 4.4 ㎍) 0 Parking, Parking on the second and fourth week New Zealand White (New Zealand White ) was vaccinated intramuscularly in the rabbit, the serum samples were collected every two weeks after challenge. IgG measurements using ELISA with respect to the collected serum are shown in Table 1. This will be described in detail as follows.
[170]
2-1. Serotype-specific IgG concentrations measured
[171]
The capsular polysaccharide for each serotype for a total of 13 kinds in 5 ㎍ / well were treated in a 96-well plate was coated for 16 hours at room temperature. Non-specific antigen of the serum in order to minimize antibody response were pooled (pooling) between the same group by taking each of the objects by serum in the same amount. Serum pool (serum pool) the C-PS (Cell wall polysaccharide; SSI (staten Serum Institute)) 333.3 ㎍ / mL and serotype 22F of the capsular polysaccharide (PnPs22F) 333.3 was adsorbed by 30 min at ㎍ / mL and room temperature , Tween 20 antibody dilution buffer to an appropriate dilution (1: 100 to 1: 40000 or so) for containing the diluted with. Washed four times for cleaning a coating buffer and the plate, the wells coated with the serum 50 ㎕ pre-adsorption and dilution-was stirred at the room temperature for 1 hour and then placed in a plate. The reaction was well-washed four times the plate in the same way, and that to each well and fixes this anti-rabbit IgG-HRP conjugate (goat anti-Rabbit IgG-HRP conjugates) (1: 20000) to insert after 30 minutes of reaction at room temperature It was. Washed 4 times in the same way as above the plate and TMB substrate solution was stabilized at room temperature, each well; Insert a (3,3 ', 5,5'-Tetramethylbenzidine Sigma, St. Louis, MO, USA) by 100 ㎕ room temperature in the reaction was carried out for 15 minutes. After putting 1 N sulfuric acid solution (Sulfuric acid solution) of 100 ㎕ to stop the reaction to control the wavelength of 650 nm was measured for absorbance at 450 nm.
[172]
It is shown in Table 1 below and the results.
[173]
TABLE 1
LBVE013 Prevenar13®
Serotype IgG(㎍/mL) Serotype IgG(㎍/mL)
19F 82.4 6A 46.5
6A 64.7 6B 22.4
14 61.1 23F 18.3
6B 59.5 19F 17.6
19A 26.7 19A 17.2
23F 21.8 14 10.1
7F 13.4 5 8.4
5 11.4 4 6.7
4 9.1 7F 6.3
1 5.3 18C 4.4
18C 4.7 3 2.7
3 3.0 1 1.6
19F 82.4 6A 46.5

[174]
As shown in Table 1, polyol (13 a) and typing serum immunogenic response pattern of the Streptococcus pneumoniae vaccine composition (LBVE013) can determine that there is a difference between the definite and the Prevenar 13® pattern. 13 in this embodiment is a vaccine composition (LBVE013) in rabbits inoculated at all serotypes Prevenar 13® than the titer of higher than Prevenar 13® was confirmed in accordance with, in particular, serotypes 1, 6B, 7F, 9V in 14 and 19F it shows an excellent effect of 2-6 times the Prevenar 13®.
[175]
2-2. Functional immunogenic Identification ( Opsonophagocytic assay, OPA )
[176]
By carrying out the OPA analysis on sera obtained from the rabbits it was evaluated for the function of the antibodies induced by typing serum.
[177]
Specifically, they were pooled (pooling) between the same group of serum taking the same amount of the serum of each individual. Streptococcus pneumoniae (Streptococcus Pneumoniae) were incubated in THY medium (Todd-Hewitt Broth w / 2% Yeast Extract) for each serotype, diluted with a buffer so that the Opsonization of 200 to 300 CFU / 10 ㎕. Mixing a Streptococcus pneumoniae 10 ㎕ diluted with a serum dilution ㎕ 20 and reacted at room temperature for 30 minutes. Then a mixed solution of complement and HL-60 cells were pre-differentiation (cells: Complement = 4: 1) was added in and the reaction was carried out for 50 ㎕ and 45 minutes at a CO 2 incubator (37 ℃).
[178]
The temperature was lowered to stop phagocytosis and smear a reaction solution 10 ㎕ in advance for 30 to 60 minutes dried agar medium. Then, CO 2 culture 12 to 18 hours in an incubator (37 ℃), and counted the number of clusters. OPA station were going to represent 50% of apoptosis which is a multiple dilution is observed.
[179]
It is shown in Table 2 below the results obtained:
[180]
TABLE 2
Serotype OPA titer LBVE013/Prevenar-13
LBVE013 Prevenar13
1 208 44 4.7
3 218 190 1.1
4 1775 836 2.1
5 493 228 2.2
6A 1606 1375 1.2
6B 1751 784 2.2
7F 2187 2187 1.0
9V 1032 512 2.0
14 2187 1761 1.2
18C 1222 1035 1.2
19A 2187 1468 1.5
19F 2187 847 2.6
23F 752 320 2.4

[181]
In Table 2, for example, it means having a very high potency in failure to reach the 50% level compared to the negative control group in the interval is the most diluted OPA titer is indicated by 2187. Through the test results, multivalent pneumococcal vaccine compositions according to the present embodiment was confirmed as compared with Prevenar 13® has a remarkably excellent serum IgG titers. Thus, the multivalent Streptococcus pneumoniae vaccine composition according to the present embodiment may be very useful in preventing a disease caused by Streptococcus pneumoniae.
[182]
See example : vaccine Room buoyancy test protocols and standards
[183]
In this embodiment, the vaccines were tested according to the standard of the room buoyancy EP-B standards requiring the vaccine by the World Health Organization (WHO) from the United States Pharmacopeia (USP) and European Pharmacopoeia (EP).
[184]
Referring to the test information, specifically, bacterial two kinds of Pseudomonas aeruginosa (ATCC number 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), a yeast Candida albicans (ATCC No. 10231, CA), fungi Aspergillus niger (ATCC No. 16404, aN) 10 in the 0th time for four kinds of vaccine composition for each of the bacteria in 5 to 10 6 CFU / mL (CFU; a Colony forming units) were inoculated. After 24 hours, 7, 14, sampled on day 28 and the number of colonies was counted in 3 to 5 days in culture in solid medium.
[185]
EP-B to the buoyancy chamber and the reference of each country pharmacopoeia of the results according to the method shown in Table 3 below.
[186]
TABLE 3
Pharmacopoeia standards Log Reduction (log CFU/mL 감소)
6 hours 24 hours 7 days 14 days 28 days
Germ EP-A 2 3 NO *
EP-B - 1 3 - NI **
USP - - 1 3 NI
JP - - - 3 NI
Yeast and fungi EP-A - - 2 - NI
EP-B - - - 1 NI
USP - - NI NI NI
JP - - - NI NI

[187]
*NR: No Recovery**NI: No Increase
[188]
As can be seen from Table 3, EP requirements of the US Pharmacopoeia (USP) or stringent than the Japanese Pharmacopoeia (JP) and is divided according to the formulation as a category A and B is the level required for vaccine production by the WHO is EP-B (EP 5.1.3. Efficacy of antimicrobial perserbation, USP 37-51, Antimicrobial effectiveness testing).
[189]
In this embodiment, multivalent pneumococcal polysaccharide-protein in developing conjugate vaccine, was added to the vaccine preservative in chimerosal and 2-PE which is generally used in the art, embodiments of the room buoyancy test, chimerosal or 2-PE in low concentrations When used alone, it was confirmed that the room does not meet the criteria buoyancy.
[190]
In addition, if the use of 2-PE (7mg / mL or more) of high concentration could not already in its application to a patent from a third-party use.
[191]
The inventors of the present invention are the experiments to develop a new composition capable of approaching pneumoniae without a preservative for protein conjugate vaccine without prejudice to the patent of others in order to satisfy the room buoyancy criteria while minimizing the amount of 2-PE increase the preservative effect the results were obtained as to the result of progress.
[192]
Rooms buoyancy screening of a protein conjugate vaccine - polyvalent pneumococcal polysaccharide: Example 3
[193]
Bacteria two kinds of Pseudomonas aeruginosa (ATCC No. 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), the yeast Candida albicans (ATCC number 10231, CA), fungi Aspergillus niger of four species of bacteria (ATCC No. 16404, AN) , well-known as well that do not die in 2-PE Staphylococcus aureus was selected (ATCC No. 6538 SA,) in bacteria for screening room buoyancy. By evaluating the buoyancy chamber in a sampling samples at 24 hours it was carried out to select the 2-PE in combination with formaldehyde for the purpose of satisfying the EP-B. Thus, the test was carried out according to the test method of EP-B.
[194]
More specifically, Example 1 multivalent pneumococcal polysaccharide-protein conjugate vaccine with reference to the manufacturing method, as shown in the following table 2 chimerosal, or 2-PE and / or the addition of formaldehyde to multivalent vaccine compositions 1 to 12 pneumococcal protein bond prepare a vaccine composition, and a preparation of a Staphylococcus aureus (ATCC No. 6538, SA) with the bacteria at 0 10 hours after 5 to 10 6, the CFU / mL (CFU = colony forming units) were inoculated. After sampling the 0 hour, at 24 hours by counting the number of colonies in the 3 to 5 days in culture in solid medium were calculated Log reduction colonies. The results are shown in Table 4.
[195]
TABLE 4
2-PE concentrations (mg / mL) Formaldehyde concentration (㎍ / mL) Chimerosal concentration (㎍ / mL) SA buoyancy chamber (24) EP-B standard
A vaccine composition 1 - - 85 Fail
A vaccine composition 2 100 Fail
A vaccine composition 3 5.0 - - Fail
A vaccine composition 4 5.0 10 - Fail
A vaccine composition 5 5.0 25 - Fail
A vaccine composition 6 5.0 40 - Fail
A vaccine composition 7 5.0 50 - Fail
A vaccine composition 8 5.0 80 - Fail
A vaccine composition 9 5.0 100 - Pass
A vaccine composition 10 5.0 120 - Pass
11. A vaccine composition 5.0 170 - Pass

[196]
As can be seen from Table 4, the 2-PE 5.0 mg / mL and formaldehyde from the combination of 100 ㎍ / mL SA room buoyancy passed the EP-B standard. The Streptococcus pneumoniae polysaccharide approach based on the results - may be seen that as a preservative for protein conjugate vaccine is 2-PE of about 5.0 mg / mL and a degree of formaldehyde above 100 ㎍ / mL in combination as appropriate.
[197]
Example 4: polyvalent pneumococcal polysaccharide-protein conjugate vaccine Room buoyancy test 1
[198]
Room buoyancy test was performed in accordance with the test method of EP-B. Vaccine compositions 13 to 15 bacteria polyvalent pneumococcal protein conjugate vaccine preparation two of Pseudomonas aeruginosa (ATCC number 9027, PA), Staphylococcus aureus (ATCC No. 6538, SA), a yeast Candida albicans (ATCC No. 10231, CA), fungi Aspergillus niger (ATCC No. 16404, aN) 10 in the 0th time four types of bacteria, each of 5 to 10 6, the CFU / mL (CFU = colony forming units) were inoculated. Since bacteria was 0 hours, 24 hours, 7 days and sampled on day 28 to count the number of colonies in the 3 to 5 days in culture in solid medium were calculated Log reduction colonies. Fungal and yeast is 0 hours, 14 days, and sampled on day 28 to count the number of colonies in the 3 to 5 days in culture in solid medium were calculated Log reduction colonies. The results are shown in Table 5.
[199]
Table 5
Sampling time Log Reduction (log CFU/mL 감소)
Germ Fungi and yeast
P.A. S.A. EP-B standard C.A. A.N. EP-B standard
A vaccine composition 13:13 the (2-PE 5.1 ​​mg / mL + formaldehyde 102 ㎍ / mL) 24 hours 1.7 2.2 1 or more - - -
7 days NO NO 3 or more - - -
14 days - - - NO 1.2 1 or more
28 days NO NO NI NO NO NI
A vaccine composition 14:15 the A (2-PE 5.1 ​​mg / mL + formaldehyde 102 ㎍ / mL) 24 hours NO 2.1 1 or more - - -
7 days NO NO 3 or more - - -
14 days - - - NO NO 1 or more
28 days NO NO NI NO NO NI
A vaccine composition 15:15 the B (2-PE 5.1 ​​mg / mL + formaldehyde 102 ㎍ / mL) 24 hours NO 1.9 1 or more - - -
7 days NO NO 3 or more - - -
14 days - - - NO NO 1 or more
28 days NO NO NI NO NO NI

[200]
*NR: No Recovery**NI: No Increase
[201]
As shown in Table 5, 13 a, 15 A and 15, each pneumococcal polysaccharide approaching, including serotypes of the B - protein conjugate vaccine compositions 13 to 15, bacterial two kinds of Pseudomonas aeruginosa (ATCC number 9027, PA) , Staphylococcus aureus (ATCC No. 6538, SA), the yeast Candida albicans (ATCC number 10231, CA), fungi Aspergillus niger results confirm the room buoyancy of the bacteria of the four species (ATCC number 16404, aN), irrespective of the serotype 13 a, 15 have satisfied the room buoyancy EP-B based on the four types of bacteria in both a and B is 15. Pneumococcal polysaccharide approach as a result - if the preservative of the protein conjugate vaccine includes a 2-PE from about 5 mg / mL and formaldehyde in an amount of about 100 ㎍ / mL was confirmed that satisfies a room buoyancy of EP-B standard .
[202]
If the art the that will typically added to a pneumococcal protein conjugate vaccine is 2-PE 7 mg / mL or more in an amount of the result of at least 100 ㎍ / mL of formaldehyde with the results of Example 3, for use as an antiseptic, 2 if you lower -PE content to 5 mg / mL shows that it is possible to achieve a room buoyancy of EP-B. Thus, the multivalent Streptococcus pneumoniae polysaccharide in the present specification - to 2-phenoxyethanol (2-Phenoxyethanol, 2-PE) and formaldehyde powerful and effective applying the optimized combination of the room (Formaldehyde, HCHO) buoyancy as a preservative of the protein conjugate vaccine multidose able to provide a dose of the composition to maintain.
[203]
Example 5: polyvalent pneumococcal polysaccharide-protein conjugate vaccine compositions stability
[204]
Example 4 multivalent pneumococcal polysaccharide in a new composition based on the results - the protein conjugate vaccine composition 16 was prepared. Specifically, a vaccine composition 16 was prepared by the following method: of 14 polysaccharide-protein conjugate (Type 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F ) and into the phosphate aluminum (aluminum phosphate) 1mg / mL (based on aluminum concentration) and the mixture was stirred to mix well. Here week 5 mM succinate and 0.85% in distilled water (w / v) and mixed with the inactivating filter having a buffer dissolved sodium chloride. A 2-PE in the final 6.0 mg / mL concentration of formaldehyde was adjusted to a final pH of 5.8 gave after homogenisation was added to 120 ㎍ / mL concentration. Thus, the concentration of each serotype in the manufacture vaccine composition is 4.4 ㎍ / mL (stage 6B is 8.8㎍ / mL) is.
[205]
In that the manufacture of a vaccine composition 16 to 2 to from 8 ℃ 6 month of storage to the content recovery of a result of the test the content of the preservative 2-PE and formaldehyde stabilized at 90 to 110% range without the loss of preservatives maintained after confirmed.
[206]
Summarizing the above results, the combination of 2-PE and formaldehyde illustrates an embodiment of the present invention polyvalent pneumococcal polysaccharide - is an excellent composition which can maintain effective long-term room buoyancy of the protein conjugate vaccine, a polyvalent polysaccharide-protein bond improved room buoyancy of the vaccine, or it was confirmed that the room buoyancy sustain period is elongated multidose be useful to provide a dosage composition.
[207]
From the above description, those skilled in the art will appreciate that may be embodied in other specific forms of the present invention without changing the technical spirit or essential characteristics. The embodiments described above In this regard, the examples should be understood as illustrative and not be limiting in all aspects. The scope of the invention should be construed as the meaning and scope, and all such modifications as derived from the equivalent concepts of the claims to be described later, rather than the description above within the scope of the invention.

Claims

[Claim 1](i) capsular polysaccharide-protein conjugate handling, (ii) 2- phenoxyethanol (2-Phenoxyethanol, 2-PE ) 4mg / mL at least 7mg / mL or less, and (iii) formaldehyde (Formaldehyde, HCHO) 90 to 200 ㎍ / mL, and including, the capsular polysaccharide is 13 to 24 kinds of Streptococcus pneumoniae (in Streptococcus pneumoniae ) which comprises capsular polysaccharides derived from serotypes, multivalent pneumococcal vaccine compositions.
[Claim 2]
The method of claim 1, wherein the capsular polysaccharide is Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F , 18C, 19A, which comprises 19F, 20, 22F, 23F, 33F, and 13 to 24 capsular polysaccharides derived from one selected from polyvalent pneumococcal vaccine compositions.
[Claim 3]
The method of claim 2 wherein the capsular polysaccharide is Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and capsular 13 kinds derived from 23F polysaccharide; Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 14 kinds of capsular polysaccharides derived from 23F; Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 15 kinds of capsular polysaccharides derived from 23F, 2, and 12F; Streptococcus pneumoniae serotypes 1, 3, 4, 15 kinds of capsular polysaccharides derived from 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 2, and 15B; Or Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, the 17 kinds of capsular polysaccharides derived from 19F, 22F, 23F and 33F which comprises polyvalent pneumococcal vaccine compositions.
[Claim 4]
The method of claim 1, wherein the transport protein is CRM197 protein, multivalent pneumococcal vaccine compositions.
[Claim 5]
The method of claim 1, wherein the capsular polysaccharide-protein conjugate is carried cyano carbonylation method of capsular polysaccharide and protein transport using this, to obtain a connected structure group -OC (NH) -NH-, multivalent pneumococcal vaccine compositions.
[Claim 6]
The method of claim 5, wherein the cyano carbonylation method, CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate) or would be performed using CNBr, multivalent pneumococcal vaccine compositions.
[Claim 7]
The method of claim 1, wherein with respect to the capsular polysaccharide 1 part by weight of serotype 1 pedigree of serotypes 2, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 23F, 12F and 15B derived from capsular polysaccharide is 0.9 to 1.1 parts by weight, and serotype 6B capsular polysaccharides derived from 1.8 to, multivalent pneumococcal vaccine composition containing 2.2 parts by weight.
[Claim 8]
Claim 1 to claim 7 as claimed in any one of claims, wherein the polyhydric pneumonia in multidose amount aureus vaccine composition.
[Claim 9]
Claim 1 to claim 7, wherein in any one of the multivalent pneumococcal vaccine composition against Streptococcus pneumoniae infection or preventing or treating Streptococcus pneumoniae infection in a pharmaceutical composition for diseases including.
[Claim 10]
10. The method of claim 9, wherein the Streptococcus pneumoniae infection is pneumonia of pharmaceutical composition.
[Claim 11]
(1) Streptococcus pneumoniae ( Streptococcus pneumoniae preparing a protein conjugate, and (2), wherein the capsular polysaccharide-capsular polysaccharides (capsular polysaccharide) of) the protein conjugates, 2-phenoxyethanol (2-Phenoxyethanol, 2 -PE) and formaldehyde (formaldehyde, is to the capsular polysaccharide comprising the step of mixing the HCHO), and comprises a 13 to 24 kinds of Streptococcus pneumoniae capsular polysaccharides derived from serotypes, of the 2-phenoxyethanol the amount is the amount of 4mg / mL and to less than 7mg / mL, formaldehyde is from 90 to 200㎍ / mL of polyvalent pneumococcal method of producing a vaccine composition.
[Claim 12]
12. The method of claim 11, wherein (1) capsular polysaccharides-for preparing a protein conjugate comprising the step of connecting via a carbonylation cyano (cyanylation) the capsular polysaccharide and the protein to perform a method -OC (NH) -NH- It is a polyvalent pneumococcal vaccine manufacturing method of composition.