Pneumococcal polysaccharides and their use in immunogenic polysaccharide-carrier protein conjugates - Patent Application 20070123333

Abstract

To provide purified capsular polysaccharides from Streptococcus pneumoniae serotype 24F, and polysaccharide-protein conjugates having this serotype.SOLUTION: The present invention provides a purified polysaccharide comprising a repeating unit with the structure below. The present invention also provides a polysaccharide-protein conjugate comprising a polysaccharide that comprises a repeating unit with the structure below and is conjugated to a carrier protein.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to a method for the treatment of Streptococcus pneumoniae. Purified capsular polysaccharide from Bacillus umoniae serotype 24F and a polysaccharide from this serotype The present invention provides a polysaccharide-protein conjugate having a glycoprotein derived from this serotype. The protein conjugate may be included in a multivalent pneumococcal conjugate vaccine. [Background technology]

[0002] Streptococcus pneumoniae, an example of an encapsulated bacterium, is a serious cause of infection worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) announced that in the United States: Each year there are 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, and 7,000 cases of ,000 cases of pneumococcal otitis media and 500,000 cases of pneumococcal pneumonia The Centers for Disease Control and Prevention event,MMWR Morb Mortal Wkly Rep 1997, 46(RR-8):1-13. Furthermore, the complications of these diseases are significant. In some studies, pneumococcal meningitis can result in mortality of up to 8% and severe neurological sequelae. The incidence of sequelae has been reported to be 25%. Arditi et al., 1998, Ped See iatrics 102:1087-97.

[0003] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, is used by adults, especially the elderly and It has been shown to be extremely beneficial in preventing pneumococcal disease in individuals at high risk of developing pneumonia. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T cell-independent immunogens that induce weak or absent responses in infants. Chemical conjugation of bacterial polysaccharide immunogens to carrier proteins improves immune responses in infants. Diphtheria toxoid (a chemically detoxified version of DT) changes the immune response to T-cell-dependent responses. DTx and CRM197 have T cell stimulating epitopes in their amino acid sequences. It has been described as a carrier protein for bacterial polysaccharide immunogens in order to enhance the binding of saccharides to the host.

[0004] The pneumococcal conjugate vaccine, Prevnar®, was then used in infants and toddlers. The seven most frequently isolated serotypes (4, 6 B, 9V, 14, 18C, 19F and 23F) and was approved by the U.S. Following the universal use of Prevnar® in the United States, Serotypes present in Revnar® significantly reduce invasive pneumococcal disease in children The Centers for Disease Control and Prevention intervention,MMWR Morb Mortal Wkly Rep 2005,5 4(36):893-7, except in certain parts of the world, where Prevnar Serotype coverage by HIV-1 is limited, and certain serotypes (e.g., 1 There is some evidence that new species (e.g., 9A) are emerging. O'Brien t al.,2004,Am J Epidemiol 159:634-44;Wh tney et al.,2003,N Engl J Med 348:1737-4 6;Kyaw et al.,2006,N Engl J Med 354:1455 -63;Hicks et al.,2007,J Infect Dis 196:1 346-54;Traore et al.,2009,Clin Infect Di See s 48:S181-S189.

[0005] Prevnar 13® is a serotype inhibitor of serotypes 1, 3, 4, 5, 6A, 6B, 7F, and 9V. 13-valent pneumococcal polysaccharide-protein complex containing 14, 18C, 19A, 19F and 23F Conjugate vaccines. See, for example, U.S. Patent Application Publication No. 2006 / 022838 0A1, Prymula et al., 2006, Lancet 367:740-4 8, and 48 in Washington, DC on October 25-28, 2008. th Annual ICAAC / ISDA 46 th Kie announced at the Annual Meeting ninger et al.,Safety and Immunologic Non -inferiority of 13-valent Pneumococcal C onjugate Vaccine Compared to 7-valent Pn eumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Tod See also Dagan et al., 1998, Infect. Immun.66:2093-2098 and Fattom,1999,Vacc See ine 17:126.

[0006] S. pneumoniae has been classified into over 90 serotypes based on the structure of the capsular polysaccharide. A list of known pneumococcal capsular polysaccharide structures is available in Geno, 2015, Clinical Mitology. Crobiology Reviews 28:871-899.

[0007] Current multivalent pneumococcal conjugate vaccines contain multiple serotypes related to the serotypes present in the vaccine. However, it is effective in reducing the incidence of pneumococcal disease caused by the pneumococcus virus. The prevalence of pneumococci expressing different serotypes is increasing. To address this issue, newly emerging pneumococcal serotypes need to be identified and characterized. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent Application Publication No. 2006 / 0228380A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997,46(RR-8):1-13 [Non-Patent Document 2] Arditi et al., 1998, Pediatrics 102:1087-97 [Non-Patent Document 3] Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005,54(36):893-7 [Non-Patent Document 4] O'Brien et al., 2004, Am J Epidemiol 159:634-44 [Non-Patent Document 5] Whitney et al.,2003,N Engl J Med 348:1737-46 [Non-Patent Document 6] Kyaw et al.,2006,N Engl J Med 354:1455-63 [Non-Patent Document 7] Hicks et al.,2007,J Infect Dis 196:1346-54 [Non-Patent Document 8] Traore et al., 2009, Clin Infect Dis 48:S181-S189 [Non-Patent Document 9] Prymula et al.,2006,Lancet 367:740-48 [Non-Patent Document 10] Kieninger et al.,Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers [Non-Patent Document 11] Dagan et al.,1998,Infect Immun.66:2093-2098 and Fattom,1999,Vaccine 17:126 [Non-Patent Document 12] Geno,2015,Clinical Microbiology Reviews 28:871-899 Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention relates to a purified capsular polysaccharide from Streptococcus pneumoniae serotype 24F. The present invention provides a method for the preparation of a serotype ... It is based in part on the structural identification of the capsular polysaccharide from this serotype. [Means for solving the problem]

[0011] Thus, in one embodiment, the present invention provides a polysaccharide having the repeating unit: R: [ka]

[0012] The polysaccharide from Streptococcus pneumoniae serotype 24F is represented by And, [ka] In the formula, n represents the number of repeating units.

[0013] In certain embodiments, the polysaccharide has between 5 and 5000 repeating units. , polysaccharides are 10-3,000, 20-2,000, 50-1,500 or 100-1 It has 1,000 repeating units.

[0014] In certain embodiments, the polysaccharide has a molecular weight of between 5 kDa and 5,000 kDa. In this embodiment, the polysaccharide is 10 kDa to 2,000 kDa, 50 kDa to 1,500 kDa. Or has a molecular weight of 100 kDa to 1,000 kDa.

[0015] The invention further provides an activated polysaccharide produced from any of the above embodiments, In this procedure, polysaccharides are activated by chemical reagents and then attached to linker or carrier proteins. In certain embodiments, activation generates a reactive group for conjugation. In certain embodiments, the polysaccharide is activated by periodate. In a particular aspect of this embodiment, activation is achieved by activating the carbon atom of arabinitol-1-PO4. This occurs in elementary 2nd place.

[0016] The present invention further relates to a method for preparing a polysaccharide or an activated polysaccharide comprising administering to a carrier protein a In a particular embodiment, a polysaccharide-protein conjugate is provided. The carrier protein is CRM197, diphtheria toxin fragment B (DTFB). , DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT Fragment C, Pertussis Toxoid, Cholera Toxoid, E.coli LT, E.co li ST, and Pseudomonas aeruginosa In a particular embodiment, the carrier protein is selected from exotoxin A from Bacillus subtilis. is CRM197.

[0017] In certain embodiments, the polysaccharide-protein conjugate is soluble in water under aqueous conditions or in dimethylformamide. Using reductive amination chemistry in aprotic solvents such as sulfoxide (DMSO) In certain embodiments, the polysaccharide-protein conjugate is prepared by reducing in DMSO. The compounds are prepared using selective amination chemistry.

[0018] In one embodiment, the present invention provides a non- Conjugated polysaccharides or polysaccharide-protein conjugates and Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F , 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23 B, 23F, 24B, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and and 38. In one subembodiment, a multivalent immunogenic composition is provided comprising a conjugate. The immunogenic composition may be a non-conjugated polysaccharide or a polysaccharide-carrier protein conjugate. In one subembodiment, the multivalent immunogenic composition comprises a non-covalent antibody, but not both. The conjugates include mixtures of polysaccharide or polysaccharide-carrier protein conjugates. In certain subembodiments, the multivalent immunogenic compositions of the invention comprise up to 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 4 Have up to 5, 50, 55, 60, 65, 70, 75, 80, 85 or 90 serotypes . [Brief description of the drawings]

[0019] [Figure 1A] 1 shows a pictorial representation of the repeating unit structure of the S. pneumoniae serotype 24F polysaccharide. [Figure 1B] A pictorial representation of the periodate-activated S. pneumoniae serotype 24F repeat structure is shown. Note that the structure shown in FIG. 1B shows the major activation sites (as activated sugars). Within the polysaccharide chain, it is not necessary for every repeat unit to be activated as shown in FIG. 1B. [Diagram 2]Figure 1 shows a 600 MHz 1D 1H NMR spectrum of capsular polysaccharide from S. pneumoniae serotype 24F in DO at 50°C. Signals arising from internal standards (DMSO and DSS-d6), residual water (HOD), and other residual components from the purification process (ethanol (EtOH), isopropanol (IPA) and acetate) are shown. Minor signals, labeled with *, are due to S. pneumoniae cell wall residues such as C-polysaccharide and / or peptidoglycan. [Diagram 3] FIG. 1 shows the one-dimensional (1D) 1H NMR identity regions used for serotype identification of S. pneumoniae serotype 24F. The signal positions of each anomeric proton of the repeating units from each monosaccharide residue are shown. [Figure 4] FIG. 1 shows a partial two-dimensional (2D) 1H-13C multiple bond correlation NMR spectrum of S. pneumoniae serotype 24F, which establishes the covalent bonds between the repeating sugar residues. The correlations that establish the glycosidic bonds are indicated in the figure. [Diagram 5] FIG. 1 shows the establishment of phosphodiester bonds within the capsular polysaccharide repeat unit of S. pneumoniae serotype 24F. [Figure 6] Top spectrum: 1D TOCSY of activated serotype 24F polysaccharide showing excitation centered at the 5.19 ppm peak with a single correlation to the 3.86 ppm signal. Bottom spectrum: standard 1D proton spectrum of serotype 24F, identifying the triplet at 5.19 ppm. [Figure 7] The top spectrum shows 1D TOCSY of the activated serotype 24F polysaccharide, with excitation centered around the signal at 5.19 ppm. The bottom figure is the multiplicity-edited gHSQCAD. The black ovals are methyl and methine groups, and the white ovals are methylene. [Figure 8] ELISA IgG antibody titers (post-dose 2) of rabbits immunized with S. pneumoniae monovalent serovars conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA) are shown. Error bars represent geometric mean + 95% confidence interval. [Figure 9]Serotype-specific OPA titers (post dose 2) from rabbits immunized with S. pneumoniae monovalent serotypes conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA) are shown. Error bars represent geometric mean + 95% confidence interval. [Figure 10] Figure 1 shows protection of C57BL / 6 mice immunized with 24F-CRM197 monovalent conjugate vaccine formulated in aluminum phosphate adjuvant (APA) from intratracheal challenge with S. pneumoniae serotype 24F. Mantel Cox log-rank test indicates that C57BL / 6 mice immunized with 24F-CRM197 / APA vaccine were significantly protected from challenge compared to both APA-immunized mice (P=0.0004) and naive mice (P<0.0001). [Figure 11] Figure 1 shows protection of CD-1 mice immunized with 24F-CRM197 monovalent conjugate vaccine formulated with aluminum phosphate adjuvant (APA) from intratracheal challenge with S. pneumoniae serotype 24F. Mantel Cox log-rank test indicates that CD-1 mice immunized with 24F-CRM197 / APA vaccine were significantly protected from challenge compared to both APA-immunized mice (P=0.0099) and naive mice (P=0.0076). [Figure 12] Figure 1 shows protection of Swiss Webster mice immunized with 24F-CRM197 monovalent conjugate vaccine formulated in aluminum phosphate adjuvant (APA) from intratracheal challenge with S. pneumoniae serotype 24F. Mantel Cox log-rank test indicates that Swiss Webster mice immunized with 24F-CRM197 / APA vaccine were significantly protected from challenge compared to both APA-immunized mice (P<0.0001) and naive mice (P=0.0002). [Figure 13]Serotype-specific (S. pneumoniae serotypes 16F, 23A, 23B, 24F, 31) pre-immunization, PD1, and PD2 geometric mean antibody titers of rabbits immunized with multivalent pneumococcal conjugate vaccine (2 μg / PnPs). Error bars represent two standard errors of the geometric mean titers for each serotype (X-axis). [Figure 14] Serotype-specific (S. pneumoniae serotypes 16F, 23A, 23B, 24F, 31) pre-immunization, PD1 and PD2 OPA dilution titers are shown for rabbits immunized with multivalent pneumococcal conjugate vaccine (2 μg / PnPs). Symbols indicate individual titers and error bars represent 95% confidence intervals (CI) of the geometric mean titers (GMT). *p<0.05, **p<0.01, ***p<0.001, ns=not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention is based in part on the identification of novel pneumococcal polysaccharide structure(s) by NMR techniques. The structure provided herein represents the original identification or It is believed to be the first correct identification.

[0021] S. pneumoniae serotype 24F polysaccharide was produced and purified from each of the strains. The produced (and purified) polysaccharides were used to identify individual Ps-CRM197 conjugates. S. pneumoniae serotype 24F has a unique polysaccharide, which This results in a unique conjugate production process. The resulting conjugate(s) can be It has been demonstrated to be immunogenic in clinical trials.

[0022] The term "polysaccharide" (Ps) as used herein includes, but is not limited to, "sugar", "Oligosaccharide", "polysaccharide", "liposaccharide", "liposaccharide" Lipopolysaccharide (LOS), Lipopolysaccharide (LPS), Glycosylate, Glycoconjugate "Gates," "derivatized or activated polysaccharides or oligosaccharides," and other immunological and cellular It may contain any antigenic saccharide element (or antigenic unit) commonly used in the field of bacterial vaccines. Unless otherwise specified, the names of polysaccharides used herein are IUB-I. UPAC Joint Commission on Biochemical Nom enclature (JCBM) Recommendations 1980. See CBN, 1982, J. Biol. Chem. 257:3352-3354. stomach.

[0023] As used herein, an "immunogenic composition" refers to an antibody that is capable of injecting an antibody into a host, such as a mammal, by injecting the antibody into a body fluid. Inhibiting humoral or cell-mediated, or humoral and cell-mediated, immune responses Such as bacterial capsular polysaccharides or polysaccharide-protein conjugates capable of inducing An immunogenic composition refers to a composition that contains an antigen. Furthermore, it may be useful to sensitize the host by presenting antigens to the host. can be produced to provide future protection to the immunized host. The composition may protect the host against infection by bacteria or reduce the severity of the infection, or The immunogenic composition may also be able to protect the host from death due to bacterial infection. can be used to generate polyclonal or monoclonal antibodies, The antibodies can be used to confer passive immunity to a subject. Using the immunogenic compositions in an animal efficacy model or in an opsonophagocytic injury assay Functional antibodies may be generated as measured by killing of bacteria via .

[0024] As used herein, the term "isolated" in reference to polysaccharides means isolated by centrifugation. , depth filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration and / or or using purification techniques known in the art, including the use of column chromatography. refers to the isolation of S. pneumoniae serotype-specific capsular polysaccharides from purified polysaccharides. In particular, isolated polysaccharides are those that are free of proteins, nucleic acids, and nonspecific endogenous polysaccharides (C polysaccharides). The isolated polysaccharides are 10%, 8%, 6%, 4% or 2% The isolated polysaccharides contain less than 50% protein impurities and / or nucleic acids. It contains less than 20% C polysaccharides relative to the target polysaccharides.

[0025] As used herein, the term "purified" in relation to bacterial capsular polysaccharides means: Refers to the purification of polysaccharides from cell lysates by means such as centrifugation, precipitation and ultrafiltration Generally, purified polysaccharide refers to the removal of cellular debris and DNA.

[0026] As used herein, the term "Mw" refers to weight average molecular weight, typically in Da or It is expressed in kDa. Mw is the amount of large molecules a polymer sample contains more than small molecules. The total mass of the nucleus is taken into consideration. Mw is determined by static light scattering, small angle neutron scattering, It can be determined by techniques such as X-ray scattering and sedimentation velocity.

[0027] As used herein, the term "Mn" refers to the number average molecular weight, typically in Da or k It is expressed in Da. Mn is calculated by dividing the total weight of the sample by the number of molecules in the sample, Gel permeation chromatography, Viscosity measurement by (Mark-Houwink equation), flux Colligative methods, e.g., vapor pressure osmometry, end group Mw / Mn can be determined by techniques such as molecular weight determination or proton NMR. Reflects dispersibility.

[0028] As used herein, the term "molar ratio" typically refers to the ratio to the nearest tenth or hundredth. A percentage is a percentage expressed as a decimal to the tenths place. For example, 0 or The molar ratios from 0.1 to 1.0 include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0. 7, 0.8, 0.9 or 1.0.

[0029] As used herein, the abbreviation "PnPs" refers to pneumococcal polysaccharides.

[0030] As used herein, "comprising" when used in conjunction with the immunogenic compositions of the invention The term refers to the inclusion of any other components such as adjuvants and excipients (antigen mixtures). (The polysaccharide-protein complex of the present invention is limited by the term "consisting of"). The term "consisting of" when used in conjunction with conjugate mixtures refers to those specific S. pneumoniae polysaccharide-protein conjugates and other S. pneumoniae polysaccharide-protein conjugates.

[0031] As used herein, the phrase "activated site" on a sugar refers to a site that is chemically modified. An activated site means that a molecule can react at a specific site to form a reactive group. The preferred tendency of the activator to react with the activator is taken into account.

[0032] As used herein, the phrase "activated polysaccharide" refers to a polysaccharide that has been modified to form reactive groups in the polysaccharide chain. Activated polysaccharides are polysaccharides that have been chemically modified. Activated polysaccharides are those in which all available activation sites have been chemically modified. It does not necessarily mean that the compound has been chemically modified.

[0033] As used herein, the phrase "degree of activation" on a polysaccharide chain refers to the degree of activation. It refers to the overall ratio between the number of chemical groups and the number of repeating units on the polysaccharide chain.

[0034] Unless otherwise specified, all ranges provided herein are limited by the recited lower and upper limits. This includes restrictions.

[0035] Monosaccharide analysis of S. pneumoniae serotype 24F revealed the following components: Glc, Rha, and G lcN, Rib (ribose) and Rib-ol-P (ribose phosphate) were shown (structure (No manufacturing information was available.) Kamerling, 2000, Pneumo coccal polysaccharides:a chemical view,p .81-114.In Tomasz(ed),Streptococcus pneu moniae molecular biology&mechanisms of d isease.Mary Ann Liebert,Inc.,Larchmont,N See Y. The structures identified in the Examples are consistent with previously identified sugar compositions. No structural information on serotype 24A or 24B polysaccharides for comparison is available to date. I couldn't.

[0036] Identification of the structures of these polysaccharide serotype(s) will allow the identification of unconjugated or or by incorporating them into pneumococcal vaccines as polysaccharide-protein conjugates. Conjugate vaccines containing streptococci and pneumococcal Ps have been developed in the art. These techniques are well known in the art. See, for example, U.S. Patent No. 6,248,570 and U.S. Patent No. 5,866 ,135 and U.S. Pat. No. 5,773,007.

[0037] capsular polysaccharide The capsular polysaccharides derived from Streptococcus pneumoniae from the serovar(s) of the present invention are Polysaccharides can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be prepared from bacteria. It can be isolated and prepared by known methods (see, for example, European Patent No. EP 497524 and and European Patent No. EP497525), preferably achieved using a homogenizer. To some extent, the size can be adjusted by microfluidization or chemical hydrolysis. In one embodiment, S. pneumoniae strains corresponding to each polysaccharide serotype are cultured in soy-based medium. The individual cells are then isolated by standard procedures including centrifugation, precipitation and ultrafiltration. The polysaccharides are purified. See, e.g., U.S. Patent Application Publication No. 2008 / 0286838 and U.S. See Patent No. 5,847,112. Sizing polysaccharides to reduce viscosity and / or improve the filterability of the subsequent conjugated product. Chemical hydrolysis can be carried out using acetic acid. Mechanical sizing can be carried out using high pressure homogenization. This may be done using shear.

[0038] In some embodiments, the purified polysaccharide prior to conjugation is between 5 kDa and 4 kDa. The molecular weight was determined by multi-angle light scattering (MALS) and refractive index. Calculated by size exclusion chromatography (SEC) combined with a RI detector In other such embodiments, the polysaccharide may be between 10 kDa and 4,000 kDa. , 50kDa~4,000kDa, 50kDa~3,000kDa, 50kDa~2,0 00kDa, 50kDa~1,500kDa, 50kDa~1,000kDa, 50kDa a~750kDa, 50kDa~500kDa, 100kDa~4,000kDa, 10 0kDa~3,000kDa, 5kDa~2,000kDa, 10kDa~2,000k Da, 50kDa~2,000kDa, 100kDa~2,000kDa, 100kDa ~1,500kDa, 100kDa~1,000kDa, 100kDa~750kDa, 100kDa~500kDa, 100~400kDa, 200kDa~4,000kDa , 200kDa~3,000kDa, 200kDa~2,000kDa, 200kDa~ 1,500kDa, 200kDa to 1,000kDa or 200kDa to 500kDa In certain embodiments, the polysaccharide has an average molecular weight of between 50 kDa and 5,000 kDa. In certain embodiments, the polysaccharide has a molecular weight of 10 kDa to 2,000 kDa, 50 kDa to 60 kDa, or 10 kDa to 2,000 kDa. It has a molecular weight of Da to 1,500 kDa or 75 kDa to 200 kDa.

[0039] In a particular embodiment, the S. pneumoniae serotype 24F polysaccharide has between 5 and 2,000 repeats. In certain embodiments, the polysaccharide has 15 to 1,500, 20 to 400, 30 It has from 300, 40 to 250, or 50 to 200 repeating units.

[0040] Carrier Proteins Polysaccharides from one or more of the serotypes are conjugated to a carrier protein ("Pr"). and may improve immunogenicity in children, elderly and / or immunocompromised patients. When multiple serotypes are used in a multivalent composition, they may be used with the same carrier protein or with different serotypes. Each capsular polysaccharide of the same serotype may be prepared using a carrier protein of are typically conjugated to the same carrier protein.

[0041] In a particular embodiment of the invention, CRM197 is used as the carrier protein. CRM197 is a non-toxic mutant of diphtheria toxin (DT). CRM197 carrier The protein was rendered non-toxic by a single amino acid substitution in fragment A at residue 52. In one embodiment, the CRM197 carrier protein is a mutant of DT, which is Corynebacterium diphtheriae (Co. From a culture of Rynebacterium diphtheria strain C7(β197) In another embodiment, CRM197 is isolated from the Typically, CRM197 is prepared recombinantly according to the methods described in. It is purified by a combination of ammonium sulfate precipitation and ion exchange chromatography. In some embodiments, CRM197 is Pfenex Expression Te chnology(TM) (Pfenex Inc., San Diego, CA). Using the method, Pseudomonas fluorescens It is prepared in the US at the University of California, San Gorgoniocarpus cens.

[0042] Other suitable carrier proteins include additional inactivated bacterial toxins, e.g., DT, Diphtheria Tetanus toxoid fragment B (DTFB), TT (tetanus toxoid), or TT fragment Fragment C, pertussis toxoid, cholera toxoid (see, for example, International Patent Application Publication No. W O2004 / 083251), E. coli LT (heat-labile enterotoxin syn), E. coli ST (heat-stable enterotoxin), and Pseudomonas eroderma Also included are exotoxin A from Bacillus subtilis. Also included are bacterial outer membrane proteins, e.g., outer membrane complex c(OMPC), porin, transferrin-binding protein, pneumococcal surface protein A (PspA; see International Patent Application Publication No. WO02 / 091998), pneumococcal adhesin PsaA, C5a peptide from group A or B streptococci dase, or Haemophilus influenzae enzae protein D, and pneumococcal pneumolysin (Kuo et al., 1999 5, Infect Immun 63;2706-13), e.g., in some way non-toxic dPLY-GMBS (International Patent Application Publication No. WO04 / 081 515) or dPLY-formol, PhtX, e.g., PhtA, PhtB, Fusions of PhtD, PhtE, and Pht proteins, e.g., PhtDE fusions, P htBE fusions (International Patent Application Publication No. WO01 / 98334 and International Patent Application Publication No. Other proteins, e.g., o-proteins ... Bovine serum albumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), Purified protein derivative of tuberculin (PPD), PorB (N. meningitidis (N. meningitidis), PD (Haemophilus influenzae Protein D; see, for example, European Patent No. EP0594610B), or immunological Functional equivalents, synthetic peptides (European Patent No. EP0378881 and European Patent No. EP0 427347), heat shock proteins (see International Patent Application Publication No. WO 93 / 177 12 and International Patent Application Publication No. WO94 / 03208), pertussis protein ( See International Patent Application Publication No. WO98 / 58668 and European Patent No. EP0471177. ), cytokines, lymphokines, growth factors or hormones (International Patent Application Publication No. W O91 / 01146), multiple human CD4+ T cells derived from various pathogen-derived antigens Artificial proteins containing cytoplasmic epitopes (Falugi et al., 2001, European J Immunol 31:3816-3824), e.g., N19 protein (B araldoi et al.,2004,Infect Immun 72:4884 -7), iron uptake proteins (see International Patent Application Publication No. WO01 / 72337 ), C. difficile toxin A or B (International Patent Publication No. No. WO00 / 61761), and flagellin (Ben-Yedidia et al. al., 1998, Immunol Lett 64:9) also It can be used as a protein.

[0043] As carrier proteins, other DT variants, e.g., CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218 :3838-3844); CRM9, CRM45, CRM102, CRM103 and C RM107, and Genetically Engineered Toxins, Ed:Frankel,Maecel Dekker Inc,1992 by Nichol Other mutations described by ls and Youle; Glu-148 to Asp, Gln or Ser and / or Ala158 to Gly deletion or mutation, and No. 4,709,017 or other mutations disclosed in U.S. Pat. No. 4,950,740. at least one or more of residues Lys516, Lys526, Phe530 and / or Mutation of Lys534, and U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,633. 5,673; or other mutations disclosed in U.S. Pat. No. 5,843,711. You can also use a fragment like this:

[0044] When a multivalent vaccine is used, a second carrier protein is used for one or more of the antigens. The second carrier protein is preferably non-toxic and non- A protein that is reactogenic and available in sufficient quantity and purity. Proteins may also be used to bind antigens, such as S. pneumoniae polysaccharides, to enhance the immunogenicity of the antigen. The carrier protein is conjugated or conjugated with a standard conjugation agent. In one embodiment, the conjugate is conjugated to a first carrier protein. Each ungated capsular polysaccharide was conjugated to the same second carrier protein. (e.g., each capsular polysaccharide molecule is conjugated to a single carrier protein) In another embodiment, the capsular polysaccharide is not conjugated to a first carrier protein. The body is conjugated to two or more carrier proteins (each capsular polysaccharide molecule is a single In such an embodiment, the same serotype Each capsular polysaccharide of is typically conjugated to the same carrier protein.

[0045] Conjugation Prior to conjugation, purified polysaccharides were reacted with carrier proteins to form saccharide-binding proteins. The polysaccharides may be chemically activated so that they can be reacted with other polysaccharides to form activated polysaccharides. The term "activated polysaccharide" as used refers to a polysaccharide that is conjugated to a linker or carrier protein. The term refers to a polysaccharide that has been chemically modified, as described below, to enable its synthesis. The purified polysaccharide may be conjugated to a linker. When the capsular polysaccharides are conjugated separately to a carrier protein, they form glycoconjugates. The polysaccharide conjugates are prepared by known coupling techniques. It is possible.

[0046] The invention further provides an activated polysaccharide produced from any of the above embodiments, In this procedure, polysaccharides are activated by chemical reagents and then attached to linker or carrier proteins. In certain embodiments, activation generates a reactive group for conjugation. In certain embodiments, the polysaccharide is activated by periodate. In a particular aspect of this embodiment, activation is achieved by activating the carbon atom of arabinitol-1-PO4. The structure of the periodate-activated serotype 24F polysaccharide is shown in Figure 1B. The formation of aldehydes mainly at the second carbon is due to the 2,3-carbon cleavage of vicinal diols. Selective cleavage of the diol or higher numbered diols (or aldehydes) A series of favorable oxidations at the carbons ultimately results in the formation of an acid at the second carbon of the arabinitol sugar. In certain embodiments, the present invention is surprising in that it requires activation. Derivatized polysaccharides and 50%, 60%, and 70% of the aldehyde groups (by periodate activation) Alternatively, a mixture of activated polysaccharides is provided in which more than 80% is arabinitol. In one embodiment, the activated polysaccharide has the structure shown in FIG. 1B.

[0047] In certain embodiments, the polysaccharide is attached to a linker, and the free end of the linker is an ester group. Thus, the linker can form a polysaccharide-linker intermediate that is At least one end of the linker is an ester group. The other end is a linker that is to form a polysaccharide-linker intermediate.

[0048] In certain embodiments, primary amine groups in the polysaccharide are used to attach the polysaccharide to the linker. In this case, the linker typically has ester groups at both ends. This is because the coupling involves the transfer of one ester group to the first saccharide in the polysaccharide by nucleophilic acyl substitution. This reaction allows the amide bond to be formed. The polysaccharide is then linked to the linker via a polysaccharide-linker intermediate. The ester group is a first ester group for reacting with a primary amine group in the polysaccharide and a second ester group in the carrier molecule. and a second ester group for reaction with the primary amine group of A typical linker is adipic acid N-hydroxysuccinimide diester (SID EA).

[0049] In certain embodiments, the coupling is indirect, i.e., to a linker. This can also be achieved with an additional linker used to derivatize the polysaccharide prior to The polysaccharide is attached to an additional linker using the carbonyl group at the reducing end of the polysaccharide. This coupling is carried out in two steps: (a1) coupling the carbonyl group with an additional linker; (a2) reacting the free end of the additional linker with the linker; In these embodiments, the additional linker typically comprises a primary amine at each end. Since the saccharide has a primary amine group, one of the primary amine groups can be converted to the carbonyl group of the polysaccharide by reductive amination. The reaction of the carboxyl groups in the polysaccharide allows step (a1) to occur. A primary amine group that reacts with the hydroxyl group is used. Typically, the additional linker has the same primary amine group at both ends. This reaction results in the formation of polysaccharide-linked saccharides, in which the polysaccharide is linked to an additional linker via a C-N bond. An additive linker intermediate is obtained.

[0050] In certain embodiments, different groups of the polysaccharide, particularly the carboxyl groups, are used to provide additional phosphoryl groups. Polysaccharides can be coupled to the carcinoma. This coupling is carried out in two steps: a1) reacting the group with an additional linker, and (a2) reacting the free end of the additional linker. with a linker. In this case, the additional linker is typically Since the amide has a primary amine group at the end, one of the primary amine groups can be activated by EDAC activation. Allowing step (a1) to occur by reacting with the carboxyl groups of the polysaccharide. Primary amine groups are used which react with the EDAC-activated carboxyl groups in the polysaccharide. A hydrazide group is suitable. Typically, the additional linker has the same primary amine group at both ends. This reaction allows the polysaccharide to be attached to an additional linker via an amide bond. A polysaccharide-additional linker intermediate is obtained.

[0051] In one embodiment, chemical activation of the polysaccharide followed by reductive amination to form a carrier. Conjugation to proteins is described in U.S. Pat. No. 4,365,170, U.S. Pat. Nos. 4,673,574 and 4,902,506, and U.S. Patent Application Publication No. 006 / 0228380, U.S. Patent Application Publication No. 2007 / 184072, U.S. Patent Application Publication No. 2007 / 0231340 and U.S. Patent Application Publication No. 2007 / 018407 1, and International Patent Application Publication No. WO2006 / 110381, International Patent Application Publication No. WO2008 / 079653 and International Patent Application Publication No. WO2008 / 143709 This can be achieved by the means described in the chemistry described above. Reaction with any oxidizing agent that is a primary hydroxyl group to an aldehyde, such as EMPO. (WO2104 / 097099), or periodate (sodium periodate, periodate Two vicinal salts for aldehydes, such as potassium arsenate or periodate, This reaction may involve activation of pneumococcal polysaccharides by reaction of hydroxyl groups. Random oxidation or biphenyl oxidation of primary hydroxyl groups of carbohydrates with the formation of reactive aldehyde groups. This results in random oxidative cleavage of the cyclic hydroxyl groups.

[0052] In this embodiment, coupling to the carrier protein is via lysyl groups on the protein. For example, the conjugation can be carried out by reductive amination via direct amination of The mixture of activated polysaccharide and carrier protein is reacted with cyanoborohydride in the presence of nickel. This is done by reacting with a reducing agent such as sodium. , in aqueous solution or in the presence of dimethylsulfoxide (DMSO). For example, U.S. Patent Application Publication No. US2015 / 0231270 and U.S. Patent Application Publication No. US2 See European Patent No. 011 / 0195086 as well as European Patent No. EP0471177B1. A strong reducing agent such as sodium borohydride is then added to oxidize the unreacted aldehyde. To jump.

[0053] Reductive amination involves two steps: (1) oxidation of polysaccharides to reactive aldehydes; (2) formation of imines (sulfates) between activated polysaccharides and carrier proteins; The method includes reducing the amine base to form a stable amine conjugate bond. Prior to purification, the polysaccharides may be reduced in size by mechanical (e.g., homogenization) or chemical methods. Hydrolysis may be used. Chemical hydrolysis may be carried out using acetic acid. Oxidation step may involve reaction with periodate. For purposes of the present invention, the term "periodate" includes Includes both periodate and periodic acid; the term also includes metaperiodate ( IO4 - ) and orthoperiodate (IO6 5- ), and various salts of periodate. (e.g., sodium periodate and potassium periodate). The capsular polysaccharide is then lyophilized in the presence of metaperiodate, preferably sodium periodate (Na In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate. The compound is oxidized in the presence of, preferably, periodic acid.

[0054] In one embodiment, the oxidizing agent is piperidine-N-oxy or pyrrolidine-N-oxy. The nitroxyl group is stable in the presence of an oxidant that selectively oxidizes primary hydroxyls, such as or nitroxide radical compounds (see, for example, International Patent Application Publication No. WO2006 / 023361). In the above reaction, the actual oxidant is In one embodiment, the stable nitroxyl or nitro The oxy radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one embodiment, the stable nitroxyl or nitroxide radical compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROX It has a YL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In the above, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, the oxidant is a molecule having an N-halo moiety. N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide , dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2, 4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-trione Riazinane-2,4,6-trione, diiodoisocyanuric acid and 1,3,5-triio Preferably, the compound is selected from the group consisting of 1,3,5-triazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

[0055] In certain embodiments, the oxidizing agent is 2,2,6,6-tetramethyl-1-pyridinium as a co-oxidant. TEMPO free radical and N-chlorosuccinimide (NC S) (described in International Patent Application Publication No. WO2014 / 097099). Thus, in one embodiment, glycoconjugates derived from S. pneumoniae can be prepared by the following steps: That is, a) a sugar is reacted with 2,2,6,6-tetramethyl-1-piperidinyloxy( Activated sugars were reacted with TEMPO and N-chlorosuccinimide (NCS) to form and b) coupling the activated sugar to a carrier protein containing one or more amine groups. The method can be obtained by a process comprising the step of reacting the TEM with a substrate (hereinafter, the process is referred to as "TEM"). This is called "PO / NCS reductive amination."

[0056] Optionally, the oxidation reaction may be quenched by adding a quenching agent. are vicinal diols, 1,2-amino alcohols, amino acids, glutathione, and sulfur Bisulfite, dithionite, metabisulfite, thiosulfate, phosphite Sulfite, hypophosphite or phosphorous acid (glycerol, ethylene glycol, Propane-1,2-diol, butane-1,2-diol or butane-2,3-diol The fatty acids may be selected from the group consisting of phenylalanine, ... and ascorbic acid.

[0057] The second step in the conjugation process for reductive amination is to prepare a stable conjugate. Activating polysaccharides using reducing agents to form gate bonds (also called reductive amination) The reduction of the imine (Schiff base) bond between the carboxylate and the carrier protein is the preferred reducing agent. cyanoborohydride (e.g. sodium cyanoborohydride) or borohydride In one embodiment, the reducing agent is sodium cyanoborohydride. .

[0058] In certain embodiments of the method of the present invention, the reductive amination reaction is carried out in an aprotic solvent (or In one embodiment, the reduction reaction is carried out in a mixture of aprotic solvents (DMSO ( The reaction is carried out in dimethyl sulfoxide (DMF) or dimethylformamide (DMF) solvents. If dry, DMSO or DMF solvent is used to separate the activated polysaccharide and carrier. The protein may be reconstituted. In one embodiment, the aprotic solvent is DMSO. .

[0059] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which may be advantageously It may be capped or quenched using a suitable capping or quenching agent. In one embodiment, the capping or quenching agent is sodium borohydride (N aBH4). Suitable alternatives include trimethylsilyl ether in the presence of a Brönsted or Lewis acid. Sodium triacetoxyborohydride or sodium borohydride or borohydride zinc), pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimeth- t-BuMe'PrN-BH3, benzylamine-BH3 or Amine boranes such as 5-ethyl-2-methylpyridine borane (PEMB), or hydrogen Contains boron dioxide exchange resin.

[0060] Glycoconjugates prepared using reductive amination in aprotic solvents were Commonly used in multivalent pneumococcal conjugate vaccines. Therefore, any serotype In certain embodiments of the multivalent composition where the remaining The reduction reaction of the serotype of (N-morpholino)ethanesulfonic acid), HEPES, (4-(2-hydroxyethyl) -1-Piperazineethanesulfonic acid), Bis-Tris, ADA (N-(2-acetamide ) iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) ), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-( N-morpholino)propanesulfonic acid), DIPSO(3-bis(2-hydroxyethyl )amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino N-(2-hydroxyethyl)piperazine-N -(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis( 2-Hydroxy-3-propanesulfonic acid), TEA (triethanolamine), EP PS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), bicine or HEPB, pH 6.0-8.5, 7.0-8.0 or 7.0-7. 5) is carried out.

[0061] In some embodiments, the glycoconjugates of the present invention have a molecular weight of 10 kDa to 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of 25.0 kDa. In other such embodiments, the polysaccharide has a molecular weight of from about 1,000 kDa to about 5,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 50 kDa and 1,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of 70 kDa to 900 kDa. In other such embodiments, the polysaccharide has a molecular weight of 100 kDa to 800 kDa. The body has a molecular weight of between 200 kDa and 600 kDa. , polysaccharides are 100kDa to 1,000kDa, 100kDa to 900kDa, 100k Da~800kDa, 100kDa~700kDa, 100kDa~600kDa, 10 0kDa~500kDa, 100kDa~400kDa, 100kDa~300kDa, 150kDa~1,000kDa, 150kDa~900kDa, 150kDa~800 kDa, 150kDa~700kDa, 150kDa~600kDa, 150kDa~5 00kDa, 150kDa~400kDa, 150kDa~300kDa, 200kDa ~1,000kDa, 200kDa~900kDa, 200kDa~800kDa, 20 0kDa~700kDa, 200kDa~600kDa, 200kDa~500kDa, 200kDa~400kDa, 200kDa~300, 250kDa~1,000kDa , 250kDa~900kDa, 250kDa~800kDa, 250kDa~700k Da, 250kDa~600kDa, 250kDa~500kDa, 250kDa~40 0kDa, 250kDa~350kDa, 300kDa~1,000kDa, 300kDa a~900kDa, 300kDa~800kDa, 300kDa~700kDa, 300 kDa~600kDa, 300kDa~500kDa, 300kDa~400kDa, 4 00kDa~1,000kDa, 400kDa~900kDa, 400kDa~800k Da, 400kDa~700kDa, 400kDa~600kDa or 500kDa~ It has a molecular weight of 600 kDa.

[0062] In certain embodiments, the conjugation reaction is carried out by reductive amination, Nickel is used to increase the efficiency of the jugation reaction and aid in the removal of free cyanide. Transition metals are known to form stable complexes with cyanides, and cyanohydrides are used. Reductive methylation of protein amino groups with formaldehyde by sodium borohydride It is known to improve the .1982,203:331-334;Jentoft et al.Anal Bio chem.1980,106:186-190). The addition of nickel reduced the remaining inhibitory By forming a complex with an anhydride, the consumption of the protein during conjugation is increased. This increases the number of nucleotides in the nucleoside analogues, resulting in the formation of larger, potentially more immunogenic, conjugates. .

[0063] Suitable alternative chemistries include 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. This involves the activation of sugars with diacyl cyanate (CDAP) to form cyanate esters. Therefore, the activated sugar can be attached to a carrier protein directly or via a spacer (linker) group. For example, the spacer can be attached to an amino group on a protein to provide a thiolated polysaccharide. The cystamine or cysteamine can be used as a cystine derivative, which is a maleimide-activated carrier protein. saccharides (e.g., using GMBS) or haloacetylated carrier proteins (e.g., Iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-succinimide sidyl bromoacetate or SIAB, or SIA, or SBAP) and The thioether bond obtained after the reaction of For example, the cyanate ester (which may be made by CDAP chemistry) is hexanediamine. Amino-derivatized sugars are conjugated with carbodiimides or adipic acid dihydrazide (ADH) to form carbodiimides. Carbohydrates on protein carriers using amide (e.g., EDAC or EDC) chemistry Such conjugates are conjugated to carrier proteins via the hydroxyl groups. The present application is incorporated herein by reference in its entirety. 8348 and International Patent Application Publication No. WO96 / 29094 and Chu et al. ., 1983, Infect. Immunity 40:245-256 do.

[0064] Other suitable techniques include carbodiimide, hydrazide, active ester, norbornene, p- Nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU were used. Many of these are described in International Patent Application Publication No. WO98 / 42721. The gating step involves the reaction of free hydroxyl groups of sugars with CDI (Bethell et al. l.,1979,J.Biol.Chem.254:2572-4;Hearn et al. al., 1981, J. Chromatogr. 218:509-18), and then Carbonyl groups that can be formed by reacting with proteins to form carbamate bonds. These may include reduction of the anomeric terminus to a primary hydroxyl group, Optionally, the primary hydroxyl groups can be protected / deprotected, and reacted with CDI to form the primary hydroxyl groups. The formation of a CDI carbamate intermediate and the interaction of the CDI carbamate intermediate with the protein Coupling with an amino group may be included.

[0065] Conjugation (reduction reaction and optionally capping or quenching reaction) The glycoconjugates are then purified (polysaccharide-catalyzed) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration, and Condensation / diafiltration, tangential flow filtration, ultrafiltration, precipitation / elution, column chromatography Chromatography (Ion Exchange Chromatography, Multimodal Ion Exchange Chromatography) filtration, DEAE or hydrophobic interaction chromatography) and depth filtration. See, for example, U.S. Patent No. 6,146,902. In one embodiment, glycoco The conjugates can be purified by diafiltration or ion exchange chromatography or Purified by size exclusion chromatography.

[0066] One method of characterizing the glycoconjugates of the invention is to characterize the glycoconjugates conjugated to the sugar. This is due to the number of lysine residues in the carrier protein (e.g., CRM197), This is characterized as the extent of conjugated lysine (degree of conjugation). Evidence for lysine modification of carrier proteins by covalent attachment to polysaccharides is This can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation produces a carrier protein that is used to create the conjugate material. In a preferred embodiment, the number of lysine residues recovered is reduced compared to the quality starting material. The degree of conjugation of the glycoconjugates of the present invention is 2-15, 2-13, 2 ~10, 2~8, 2~6, 2~5, 2~4, 3~15, 3~13, 3~10, 3~8, 3 ~6, 3~5, 3~4, 5~15, 5~10, 8~15, 8~12, 10~15 or 1 In one embodiment, the conjugation of the glycoconjugate of the present invention is The degree of is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 In a preferred embodiment, the glycoconjugates of the present invention have a .alpha.-amino acid residue of about 13, about 14 or about 15. The degree of conjugation of the gates is 4 to 7. In some such embodiments , the carrier protein is CRM197.

[0067] The glycoconjugates of the present invention may also be characterized by the ratio of sugar to carrier protein (wt / wt) In some embodiments, multiple glycoconjugates may be characterized by The ratio of glycoform to carrier protein (w / w) is 0.5 to 3.0 (e.g., about 0.5, about 0.6, approx. 0.7, approx. 0.8, approx. 0.9, approx. 1.0, approx. 1.1, approx. 1.2, approx. 1.3, approx. 1.4, approx. 1.5, approx. 1.6, approx. 1.7, approx. 1.8, approx. 1.9, approx. 2.0, approx. 2.1, approx. 2.2, approx. 2.3, approx. 2.4, approx. 2.5, approx. 2.6, approx. 2.7, approx. 2.8, approx. 2.9 or In another embodiment, the sugar to carrier protein ratio (w / w) is about 0.5~2.0, 0.5~1.5, 0.8~1.2, 0.5~1.0, 1.0~1.5 In a further embodiment, the ratio of sugar to carrier protein (w / w) is 0.8 to 1.2. In a preferred embodiment, the capsular polysaccharide in the conjugate In some such embodiments, the ratio of the carboxyl group to the carrier protein is 0.9 to 1.1. In this example, the carrier protein is CRM197. The immunogenic composition may be a free saccharide that is not covalently conjugated to a carrier protein. may still be present in the glycoconjugate composition. The sugars are non-covalently attached to the glycoconjugate (i.e., The glycoconjugates may be non-covalently bound, adsorbed or entrapped in or with the glycoconjugates.

[0068] In a preferred embodiment, the glycoconjugate comprises about 50% as compared to the total amount of polysaccharide; Contains less than 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides In a preferred embodiment, the glycoconjugate comprises less than about 25% of the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate contains less than about 100% free polysaccharide. In a preferred embodiment, the glycoconjugates contain less than about 20% free polysaccharides compared to the glycoconjugates. The saccharide contains less than about 15% free polysaccharides compared to the total amount of polysaccharides.

[0069] Multivalent polysaccharide-protein conjugate vaccines In a particular embodiment of the invention, to provide a multivalent pneumococcal vaccine, free polysaccharides, Streptococcus cerevisiae may be used as a component of the polysaccharide-protein conjugate or a combination thereof. Unconjugated polysaccharide or polysaccharide-protein from Cass pneumoniae serotype 24F Conjugates and S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 1 5B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A , 22F, 23A, 23B, 23F, 24B, 27, 28A, 31, 33F, 34, 35 and capsular polysaccharides derived from one or more of 35A, 35B, 35F and 38. In certain embodiments of the invention, the immunogenic composition comprises one or more carrier proteins. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, conjugated individually to proteins 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43 or 44 capsular polysaccharides from S. pneumoniae serotypes Preferably, the specific serotype-specific The incoming saccharide is not conjugated to multiple carrier proteins.

[0070] After the individual glycoconjugates are purified, they are combined to form the immunogenic composition of the invention. These pneumococcal conjugates are prepared by a separate process. , bulk formulated into a single dosage formulation.

[0071] Pharmaceutical / vaccine compositions The present invention further provides the above polysaccharides together with a pharma- ceutically acceptable carrier and adjuvant. comprising or consisting essentially of any combination of S. pneumoniae serotypes, or The present invention provides compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising:

[0072] Formulation of the polysaccharide-protein conjugates of the present invention is art-recognized. For example, the individual pneumococcal conjugates can be prepared using the method of preparing the composition. The pharmaceutical composition may be formulated with a physiologically acceptable vehicle for the administration of the pharmaceutical composition. Examples of such vehicles include , including but not limited to, water, buffered saline, polyols (e.g., glycerol, proline pyrene glycol, liquid polyethylene glycol) and dextrose solution.

[0073] In a preferred embodiment, the vaccine composition is an L-histidine buffer containing sodium chloride. It is formulated as a liquid.

[0074] As defined herein, an "adjuvant" is an agent that enhances the immunogenicity of the immunogenic composition of the present invention. Immunoadjuvants are substances that help increase the virulence of a given vaccine. Is immunogenic, e.g., does not induce any antibody titers, induces weak antibody titers, or induces cytotoxicity. Enhances immune responses to antigens that induce cystic immune responses and increases antibody titers to the antigens. and / or reduce the dose of antigen effective to achieve an immune response in an individual. Thus, adjuvants are often administered to enhance the immune response and are well known to those skilled in the art. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to, , including the following: (1) Aluminum salts (alum), for example, aluminum hydroxide, aluminum phosphate um, aluminum sulfate, etc.; (2) Certain immunostimulants, such as muramyl peptides (defined below), or bacterial cell wall components (a) Model 110Y Mycobacterium tuberculosis (with or without glycerol) Microfluidizers such as Microfluidics (Newton, MA) 5% squalene, 0.5% formulated into submicron particles using a Chlofluidizer Containing Tween 80 and 0.5% Span 85 (with various amounts of MTP-PE) (b) MF59 (International Patent Application Publication No. WO90 / 14837), which may contain Microfluidized to micron emulsions or to produce larger particle size emulsions 10% squalene, 0.4% Tween 80, 5% Pluronic acid, vortexed for SAF containing block polymer L121 and thr-MDP, (c) 2% s Allen, 0.2% Tween 80, as well as that described in U.S. Pat. No. 4,912,094. 3-O-deacylated monophosphoryl lipid A (MPL™), trehalose dimycole Detoxify (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxify). (TM) containing one or more bacterial cell wall components from the group consisting of (R.A.S.), (Corixa, Hamilton, MT); (d) Monta nide ISA; (3) Quil A or STIMULON QS-21 (Antigenic saponin adjuvants such as saponin adjuvants (e.g., U.S. Pat. No. 6,313,623, Framingham, Mass.); see US Pat. No. 5,057,540) or ISCOMs (cholesterol, saponin , an immune stimulating complex formed by the combination of a phospholipid and an amphipathic protein) and Iscomatrix® (has essentially the same structure as ISCOM, but is tamper-resistant) particles produced therefrom, such as silica (containing no carbide); (4) Available from Corixa and described in U.S. Pat. No. 6,113,918. Aminoalkyl glucosamine phosphate compounds (AGPs) or their derivatives Synthetic lipid A analogs, such as bacterial lipopolysaccharides or analogs; one such AGP is an aqueous form Also known as 529, which is formulated as a solid or stable emulsion (previously 2-[(R)-3-tetradecanoyloxytetradecane (formerly known as RC529)] 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetrahydropyranylamino]ethyl tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoyl] tradecanoylamino]-bD-glucopyranoside; (5) Synthetic polymers, such as oligonucleotides containing CpG motif(s). rinucleotides (U.S. Patent No. 6,207,646); (6) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL -4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), Interferons (e.g., gamma interferon), granulocyte-macrophage colony stimulators Macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, and (7) Complement, for example, the trimer of complement component C3d.

[0075] In another embodiment, the adjuvant is a combination of two, three or more of the above adjuvants. A mixture of these, for example, SBAS2 (3-deacylated monophosphoryl lipids A and Q S21).

[0076] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonine. N-acetyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-amyl Lanin-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl This includes MTP-PE, etc.

[0077] In certain embodiments, the adjuvant is an aluminum salt. The vaccine may be an alum-precipitated or alum-adsorbed vaccine. Mu salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. .Lane(1988;Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory) and Nickel as,W.(1992;Aluminum salts.Research in Im The aluminum salts are described in J. Municipal Chemistry 143:489-493. Non-limiting examples include hydrated alumina, alumina hydrate, and alumina trihydrate (ATH). , Aluminum Hydrate, Aluminum Trihydrate, Alhydrogel®, S uperfos, Amphogel®, Aluminum(III) Hydroxide, Hydrogen Aluminum phosphate adjuvant (APA), amorphous aluminum aluminum trihydrate or aluminum trihydroxide.

[0078] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is an aqueous suspension of aluminum chloride. Aluminum hydroxyphosphate is made by blending aluminum and sodium phosphate in a 1:1 volume ratio. After the blending process, it is mixed with a high shear mixer. The material is then size-reduced to achieve a monodisperse particle size distribution. The product is diafiltered and steam sterilized.

[0079] In certain embodiments, commercially available Al(OH)3 (e.g., Denmark / Accura te Chemical and Scientific Co.,Westbury, Proteins were extracted using Alhydrogel® or Superfos (NY) In another embodiment, protein adsorption is determined by the pI (isoelectric pH) of the protein. and the pH of the medium. Proteins with a low pI are more potent than proteins with a high pI. The aluminum salts slowly dissolve over a period of 2-3 weeks. and establish a reservoir of Ag that is released and inhibits nonspecific activation of macrophages and complement activity. may be involved in the induction of uric acid metabolism and / or stimulate innate immune mechanisms (possibly through stimulation of uric acid) For example, Lambrecht et al., 2009, Curr Opin See Immunol 21:23.

[0080] Monovalent bulk aqueous conjugates are typically blended together and diluted. Once diluted, the batch is sterile filtered. Aluminum phosphate adjuvant is aseptically added and Target a final concentration of 4 μg / mL for all S. pneumoniae serotypes except serotype 6B. This was diluted to a target value of 8 μg / mL, resulting in a final aluminum concentration of 250 μg / mL. The adjuvanted formulation batch is filled into vials or syringes.

[0081] In certain embodiments, the adjuvant comprises a CpG-containing nucleotide sequence, e.g., CpG Containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG ODNs) In another embodiment, the adjuvant is Coley Pharmaceuticals The ODN 1826 is available from the Al Group.

[0082] "CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide" "Nucleotide" and similar terms refer to a nucleotide sequence of 6 to 50 nucleotides in length that contains an unmethylated CpG moiety. For example, Wang et al., 2003, Vacci ne 21:4297. In another embodiment, the art-recognized use of this term is Any other definition given herein is intended. A CpG-containing oligonucleotide includes any combination of Modified oligonucleotides using synthetic internucleoside linkages, modified bases and / or modified sugars This includes

[0083] Methods for using CpG oligonucleotides are well known in the art, for example, Sur e t al.,1999,J Immunol.162:6284-93;Verthel yi, 2006, Methods Mol Med. 127:139-58; and Ya suda et al.,2006,Crit Rev Ther Drug Carr This is described in Citation Syst. 23:89-110.

[0084] Administration / Dosage The compositions and formulations of the present invention are useful for administering vaccines via systemic or mucosal routes. for use in preventing or treating infectious diseases, such as pneumococcal infections, in humans susceptible to such diseases. In one embodiment, the present invention provides S. pneumoniae capsular polysaccharide conjugates. A method for inducing an immune response to a medicament comprising: In another embodiment, the present invention provides a method for treating a pneumococcus pneumoniae infection comprising administering to a human an amount of the pneumococcus pneumoniae infection. A method of vaccinating a human against infection comprising administering to the human an immunological effect of an immunogenic composition of the invention. administering an effective amount to a human.

[0085] The optimal amounts of components for a particular vaccine can be determined using standard techniques, including observation of appropriate immune responses in subjects. For example, in another embodiment, the administration of human vaccination The amount is determined by extrapolating from animal studies to human data. Dosage is determined empirically.

[0086] An "effective amount" of the composition of the present invention is an amount that is sufficient to inhibit the proliferation of a microorganism, such as S. neutrophils, during a subsequent challenge. The dose required to induce antibodies that significantly reduce the likelihood or severity of Monier's infection Point.

[0087] The methods of the invention are applicable to the treatment of invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections ( Acute otitis media and sinusitis) caused by microorganisms such as S. pneumoniae The compounds may be used to prevent and / or alleviate the primary clinical syndrome caused by the

[0088] Administration of the compositions of the invention can include injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes, or or via mucosal administration to the oral / gastrointestinal, respiratory or genitourinary tract In one embodiment, intranasal administration is used to treat pneumonia or otitis media ( It is relatively effective in preventing nasopharyngeal carriage of pneumococcus and thus attenuating infection at an early stage. (This is because it is possible to do so.)

[0089] The amount of conjugate in each vaccine dose was determined to be sufficient to induce a protective immune response without significant adverse effects. Such amounts may vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose is 0.1-100 μg of each polysaccharide. For example, each dose may include 0.1 to 10 μg, more specifically 1 to 5 μg. 100, 150, 200, 250, 300, 400, 500 or 750 ng or are 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90 Alternatively, it may contain 100 μg of each polysaccharide.

[0090] The optimal amounts of components for a particular vaccine can be determined using standard techniques, including observation of appropriate immune responses in subjects. For example, in another embodiment, the administration of human vaccination The amount is determined by extrapolating from animal studies to human data. Dosage is determined empirically.

[0091] In one embodiment, the dose of aluminum salt is 10, 15, 20, 25, 30, 50, 7 0, 100, 125, 150, 200, 300, 500 or 700 μg or 1, 1 In yet another embodiment, the above Doses of aluminum salts are per μg of recombinant protein.

[0092] In general, each 0.5 mL dose contains 2 μg of each S. nucleoside except for serotype 6B polysaccharide, which is 4 μg. -monier polysaccharide, approximately 32 μg CRM197 carrier protein (e.g., 32 μg ± 5 μg, ±3μg, ±2μg or ±1μg), 0.125mg of elemental aluminum (0. 5 mg of aluminum phosphate) adjuvant, as well as sodium chloride and L-histidine. The sodium chloride concentration is about 150 mM (e.g. For example, 150 mM ± 25 mM, ± 20 mM, ± 15 mM, ± 10 mM or ± 5 mM). and about 20 mM (e.g., 20 mM ± 5 mM, ± 2.5 mM, ± 2 mM, ± 1 mM or ±0.5 mM) L-histidine buffer.

[0093] According to any of the methods of the present invention, in one embodiment, the subject is a human. In this case, the human patient may be an infant (<1 year of age), a toddler (approximately 12-24 months of age) or a young child. In another embodiment, the human patient is an elderly patient (over 65 years of age). The compositions of the present invention may also be used in older children, adolescents and adults (e.g., 18-45 years of age or older). Suitable for use by people aged 18 to 65.

[0094] In one embodiment of the method of the present invention, the composition of the present invention is administered as a single inoculation. In embodiments, the composition is administered two, three, four or more times at appropriately spaced intervals. For example, the composition may be administered at 1, 2, 3, 4, 5, or 6 month intervals, or any combination thereof. The vaccination schedule is similar to that specified for the pneumococcal vaccine. For example, in the case of invasive disease caused by S. pneumoniae, The infant routine schedules are for ages 2, 4, 6, and 12-15 months. Thus, in a preferred embodiment, the composition is administered to mice aged 2, 4, 6 and 12-15 months. It is administered as a four-dose series at 100 mg / kg / day.

[0095] The compositions of the invention may also include one or more proteins from S. pneumoniae. Examples of S. pneumoniae proteins suitable for incorporation include those described in International Patent Application Publication No. WO02 / 023662. 083855 and those identified in International Patent Application Publication No. WO02 / 053761. nothing.

[0096] formulation The compositions of the present invention can be administered parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, intradermally, or intradermally. The drug may be administered to a subject by one or more methods known to those of skill in the art, such as intravenously, intraperitoneally, or intraperitoneally. It can be formulated accordingly.

[0097] In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous injection, intra-arterial injection, or intravenous injection in a liquid formulation. The drug is administered via intravenous, subcutaneous or intrarespiratory mucosal injection. Liquid preparations for injection are available in the form of solutions, etc. Including etc.

[0098] The compositions of the invention are available in single dose vials, multi-dose vials or pre-filled syringes. The composition can be formulated as described above.

[0099] In another embodiment, the compositions of the invention are administered orally and therefore may be in a form suitable for oral administration. Solid oral dosage forms include tablets, capsules, and capsules. Liquid oral formulations include solutions, suspensions, dispersions, etc. This includes liquids, emulsions, oils, etc.

[0100] Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solutions, suspensions, emulsions, and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. , alcoholic / aqueous solutions, emulsions or suspensions, e.g., saline and buffer solutions. Examples of oils include those of animal, vegetable or synthetic origin, e.g., peanut oil. oil, soybean oil, olive oil, sunflower oil, fish liver oil, other fish oils, or derived from milk or eggs It is a lipid that comes from

[0101] Pharmaceutical compositions may be isotonic, hypotonic or hypertonic. It is often preferred that a pharmaceutical composition be essentially isotonic upon administration. The pharmaceutical composition may preferably be isotonic or hypertonic for storage. If the solution is hypertonic due to its composition, it can be diluted to an isotonic solution prior to administration.

[0102] The isotonicity agent may be an ionic isotonicity agent, such as a salt, or a non-ionic isotonicity agent, such as a carbohydrate. Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl2, KC Examples of non-ionic isotonicity agents include, but are not limited to, sucrose, glycerol, glycerol, sorbitol, sorbitol hydrate, sorbitol glycerol, sorbitol hydrate ... Includes cellulose, trehalose, mannitol, sorbitol and glycerol.

[0103] Also preferably, the at least one pharma- ceutically acceptable excipient is a buffer. For some purposes, for example when a pharmaceutical composition is meant for infusion or injection, the composition The material can buffer a solution to a pH in the range of 4-10, e.g., 5-9, e.g., 6-8. It is often desirable to include a buffer that

[0104] Buffers include, for example, Tris, acetate, glutamate, lactate, maleate, thalate, etc. Thalate, phosphate, citrate, carbonate, glycinate, L-histidine glycine, succinate and triethanolamine buffers. do.

[0105] The buffer may, for example, be a buffer solution conforming to the USP guidelines for parenteral use, particularly if the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from monobasic acids, such as acetic acid, benzoic acid, , gluconic acid, glyceric acid and lactic acid; dibasic acids, such as aconitic acid, adipic acid, Ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid, polybasic acids, e.g. For example, citric acid and phosphoric acid; and bases, for example, ammonia, diethanolamine, It may be selected from the group consisting of lysine, triethanolamine and tris.

[0106] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution. solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution Intravenous vehicles include fluid and nutrient replenishers, Ringer's dextran, and fixed oils. Examples include electrolyte replacement fluids such as those based on tallow and those based on surfactants and Such as water and oils, with or without the addition of other pharma- ceutically acceptable adjuvants. It is a sterile liquid. Generally, it is water, saline, aqueous dextrose and related sugar solutions, glucose Coal, e.g., propylene glycol or polyethylene glycol, polysorbate 80 (PS-80), polysorbate 20 (PS-20) and poloxamer 188 (P 188) are preferred liquid carriers, particularly for injectable solutions. Examples of oils include animal, vegetable or those of synthetic origin, e.g. peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, Another fish oil, or lipids derived from milk or eggs.

[0107] The formulation may also contain a surfactant. Preferred surfactants include, but are not limited to, However, polyoxyethylene sorbitan ester surfactants (commonly known as Tweens) are (particularly PS-20 and PS-80; sold under the trade name DOWFAX™) Ethylene oxide (EO), propylene oxide (PO) and / or butyl copolymers of ethylene oxide (BO), e.g., linear EO / PO block copolymers; Octoxynol, which may have a variable number of octoxy(oxy-1,2-ethanediyl) groups; Cetoxynol-9 (Triton X-100 or t-octylphenoxypolyether Of particular interest are (octylphenoxy)polyethoxyethanol (I GEPAL CA-630 / NP-40; phospholipids, such as phosphatidylcholine ( lecithin; nonylphenol ethoxylates, such as Tergitol™ NP Series; Lauryl, Cetyl, Stearyl and Oleyl Alcohols (Brij Surfactants Polyoxyethylene fatty ethers derived from fatty acids such as triethylene glycol, glycol monolauryl ether (Brij 30); and sorbitan ester ( Sorbitan trioleate (commonly known as SPAN) 85) and sorbitan monolaurate. Preferred for inclusion in emulsions The surfactant is PS-20 or PS-80.

[0108] Mixtures of surfactants can be used, e.g. PS-80 / Span 85 mixtures. Polyoxyethylene sorbitan monooleate (PS-80) Ethylene sorbitan ester and t-octylphenoxypolyethoxyethanol (Tri Combinations with octoxynol such as methyl methacrylate X-100 are also suitable. The combination is Laureth 9 + Polyoxyethylene Sorbitan Ester and / or Octoxy Including Nol.

[0109] A preferred amount of surfactant is polyoxyethylene sorbitan ester (e.g., PS-80). ) 0.01 to 1% w / v, in particular about 0.1% w / v; octyl or nonylphenoxypoly Oxyethanol (Triton X-100, or other washes in the Triton series) agents, etc.) 0.001-0.1% w / v, especially 0.005-0.02% w / v; polyoxyethylene Ethylene ether (such as Laureth 9) 0.1-20% w / v, preferably 0.1-10% w / v, in particular 0.1-1% w / v or about 0.5% w / v.

[0110] In certain embodiments, the composition contains 250 μg / mL APA (aluminum azide phosphate). L-histidine (20 mM), saline (150 mM), and p H5.8 consisting essentially of 0.2% w / v PS-20. PS-20 ranges from 0.005 to 1. The presence of PS-20 or PS-80 in the formulation may range from 0.1% w / v. Controlling aggregation during simulation and during shipping using primary packaging. The process is Up to 44 S. pneumoniae polysaccharides in L-histidine, sodium chloride and PS-20 Combining a blend of somatic serotypes and then combining the blended material with APA and and sodium chloride, with or without an antimicrobial preservative.

[0111] The choice of surfactant must be optimized for different drug products and drug substances. For multivalent vaccines containing 15 or more S. pneumoniae polysaccharide serotypes, PS- 20 and P188 are preferred. The choice of chemistry used to prepare the conjugate also determines the formulation. In particular, the stabilization of aqueous or DM Pneumococcal polysaccharide-protein conjugates prepared in SO solvent and combined into multivalent compositions Dugates exhibit significant differences in stability depending on the particular surfactant system used in the formulation.

[0112] In the formulations described herein, the poloxamer generally has a molecular weight of 1,100 Da to 17,400 Da. Da, 7,500Da to 15,000Da or 7,500Da to 10,000Da The poloxamers are either poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulation of the present invention can be selected from the range of 0.001 to 5. % w / v or 0.025-1% w / v. Poloxamer-containing surfactant systems are In certain embodiments, the polyol is propylene glycol. In certain embodiments, the polyol is a polyol having a final concentration of 1-20% w / v. Ethylene glycol 400, at a final concentration of 1-20% w / v.

[0113] Suitable polyols for the formulation include polymeric polyols, particularly but not limited to: Propylene glycol and polyethylene glycol, polyethylene glycol monomethyl Propylene glycol is a polyether diol containing propylene ether. Polyethylene glycol is available in a range of monomer molecular weights from 2,700 Da to 3,000 Da. Also included are, but are not limited to, polyethylene glycol monomethyl ether. PEG200, PEG300, PEG400, PEG1000, PEG MME 550 , PEG MME 600, PEG MME 2000, PEG MME 3350 and and PEG MME 4000, with molecular weights ranging from about 200 Da to about 35,000 Da. The preferred polyethylene glycol is polyethylene glycol The final concentration of polyol in the formulation is 1-20% w / v or 6-20% w / v. / v may be used.

[0114] The formulation also contains a pH buffered saline solution. The buffer solution may be, for example, Tris, acetate, , glutamate, lactate, maleate, tartrate, phosphate, citrate, Carbonate, glycinate, L-histidine, glycine, succinate, HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-( N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) The buffer may be selected from the group consisting of: Solutions can be buffered to pH values ​​ranging from 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In certain embodiments, phosphate, succinate, L-histidine, MES, MOP The buffer is selected from the group consisting of S, HEPES, acetate or citrate. The buffer may be, for example, a USP compatible buffer for parenteral use, particularly if the pharmaceutical formulation is for parenteral use. The concentration of the buffer may be selected from 1 mM to 50 mM or 5 mM to 50 mM. In a particular embodiment, the buffer contains L-His at a final concentration of 5 mM to 50 mM. L-cysteine ​​or succinate at a final concentration of 1 mM to 10 mM. Histidine is at a final concentration of 20 mM ± 2 mM.

[0115] Saline (i.e., a solution containing NaCl) is preferred, but other salts suitable for the formulation may be used. These include, but are not limited to, CaCl2, KCl, and MgCl2 and their salts. Combinations include, but are not limited to, sucrose, trehalose, mannitol Although non-ionic isotonic agents including sorbitol and glycerol were used instead of salt, Suitable salt ranges include, but are not limited to, 25 mM to 500 mM or 40 mM. In one embodiment, the saline solution is NaCl, and optionally 2 mM to 170 mM. It may be present at a concentration between 0 mM and 170 mM.

[0116] In a preferred embodiment, the formulation contains an L-histidine buffer with sodium chloride. .

[0117] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. It can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration. In another embodiment, for example, the polymeric material is used in a microsphere or an implant. can be.

[0118] The compositions of the invention may also include one or more proteins from S. pneumoniae. Examples of S. pneumoniae proteins suitable for incorporation include those described in International Patent Application Publication No. WO02 / 023662. 083855 and those identified in International Patent Application Publication No. WO02 / 053761. nothing.

[0119] Analysis method Molecular weight and molecular weight of the conjugates using HPSEC / UV / MALS / RI assay Concentration analysis Conjugate samples were purified by high performance size exclusion chromatography (HPSEC). Ultraviolet (UV), Multi-Angle Light Scattering (MALS) and Refractive Index (RI) Detection is achieved using sequential detectors. The extinction coefficients are used to determine the UV280 to protein concentration. Concentration is calculated by the change in refractive index of the solution with the change in solute concentration reported in mL / g. Using a certain dn / dc factor, RI signals (both by proteins and polysaccharides) The polysaccharide concentration is deconvoluted from the resulting peaks. Astra software (Wyatt Techno) using constant concentration and light scattering information The trial was conducted by logy Corporation, Santa Barbara, CA. The average molecular weight of the material is calculated. Polydisperse molecules have multiple average molecular weights. For example, the number average molecular weight Mn, the weight average molecular weight Mw and the z-average molecular weight Mz (Molecular Weight s, 2015, 20:10313-10341). Unless otherwise specified, The term "molecular weight" used throughout is the weight average molecular weight.

[0120] Conjugated saccharides as a measure of the number of covalent bonds between the polysaccharide and the carrier protein Determination of lysine consumption of proteins To measure the degree of conjugation in the conjugate samples, Waters A ccQ-Tag amino acid analysis (AAA) is used. The samples were hydrolyzed using gas phase acid hydrolysis in the presence of carrier proteins to their constituents. Decomposes into amino acids. 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate Free amino acids are derivatized using AQC. The derivatized samples were analyzed using UPLC with UV detection. Use the appropriate amino acids to obtain the average protein concentration. Lysine consumption during conjugation ( i.e., lysine loss) is the mean measured lysine content in the conjugate compared to the predicted amount in the starting protein. It is determined by the difference between the measured lysine amount.

[0121] Free polysaccharide test Free polysaccharides in the conjugate sample (i.e., not conjugated to CRM197) Polysaccharides (not included) were combined with free proteins and deoxycholate (DOC) and hydrochloric acid. The conjugate is measured by first precipitating it. The precipitate is then filtered and the filtrate is then filtered. The liquid is analyzed for free polysaccharide concentration by HPSEC / UV / MALS / RI. Polysaccharides as a percentage of total polysaccharides measured by HPSEC / UV / MALS / RI It is calculated.

[0122] Free Protein Test Capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode was used to Free polysaccharide, polysaccharide-CRM197 conjugate and free CRM in conjugate samples Briefly, samples were diluted in 25 mM borate, 100 mM SDS, pH 9. Mix the preconditioned bare molten system with MEKC running buffer containing .3. The separation was monitored at 200 nm and a CRM197 standard curve was plotted. Free protein results are analyzed using HPSEC / UV / MA. Reported as percentage of total protein content determined by LS / RI procedure.

[0123] Various embodiments of the present invention have been described with reference to the accompanying description and drawings. It is not intended to be limited to these precise embodiments, but rather to be construed as limiting the scope of the appended claims. Various changes and modifications may be made by those skilled in the art without departing from the scope or spirit of the present invention. It should be understood that this can be done.

[0124] The following examples illustrate but do not limit the invention.

[0125] [Example] [Example 1] Preparation of S. pneumoniae capsular polysaccharide Methods for culturing pneumococci are well known in the art. See, e.g., Chase, 1967 ,Methods of Immunology and Immunochemist See, for example, U.S. Pat. No. 6,233,363, and U.S. Pat. No. 6,233,363. Methods for preparing pneumococcal capsular polysaccharides are also known in the art. See, for example, European Patent No. EP0497524B1. The process generally follows the method described in European Patent No. EP0497524B1. Except as modified, these guidelines are generally applicable to all pneumococcal serotypes.

[0126] Pneumococcal subtype 24F isolates were obtained from Merck Culture Collection. ion. If necessary, the results were based on the Quelling reaction using specific antisera. Subtypes can be distinguished by using the ELISA kit. See, e.g., U.S. Patent No. 5,847,112. Contains soy peptone, yeast extract, and hemin-free glucose. The obtained strains were successively plated on agar plates made of animal component-free medium in two steps. The isolated strain was further cloned. Contains thorium, sodium bicarbonate, potassium phosphate, glucose and glycerol. Clonal isolates of each serotype were further propagated in liquid culture using animal component-free medium. A pre-master cell bank was prepared.

[0127] The production of each serotype of pneumococcal polysaccharide involves cell growth and batch production fermentation followed by downstream purification. The soybean peptone or soybean peptone ultrafiltrate, yeast eel, and yeast extract were used for the preparation of the yeast strain fermentation. Kiss or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, A shaker containing a pre-sterilized, animal component-free growth medium containing potassium phosphate and glucose Using flasks or culture bottles, transfer vials of thawed cell banks from each serotype. Cell proliferation cultures were grown in closed shake flasks or bottles at tem- perature and gas exchange was minimized by controlling stirring. Measured by optical density at 600 nm After achieving a certain culture density, soy peptone or soy peptone ultrafiltration is performed. yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate and glucose The cell growth medium was added to a production fermenter containing a pre-sterilized, animal component-free growth medium containing glucose. Part of the nutrient was transferred. Temperature, pH, pressure and agitation were controlled. No sparging was used. Airflow overlay was also controlled because

[0128] When glucose was nearly depleted, the buffer was inactivated by adding a chemical inactivator, phenol. Fermentation was terminated by adding pure phenol to a final concentration of 0.8–1.2% to dissolve the cells. The primary inactivation was performed by continuously increasing the temperature and stirring. This occurs in a fermenter that is controlled by a centrifuge for a specific period of time. After the primary inactivation, the batch is transferred to a separate vessel. There, the cells are inactivated for a specific period of time under temperature and agitation control to ensure complete inactivation. This was achieved by microbial plating techniques or by measuring phenol concentrations. This was confirmed by verification of the degree and specific time. The inactivated broth was then purified.

[0129] Purification of Ps The purification of pneumococcal polysaccharides involves several steps: centrifugation, depth filtration, and concentration / diafiltration. All steps were performed at room temperature unless otherwise stated. Ta.

[0130] Cationic polymer (BPA-1000, Petrolite "Tretolite" and "Spectrum 8160" and poly(ethyleneimine), "Millip ore pDADMAC) from fermenter cultures of S. pneumoniae. The cationic polymer flocculated the fermented broth, flocculating the impurities proteins, nucleic acids and cell debris. After the flocculation step and maturation period, the mixture was purified by centrifugation and multiple depth filtration steps. The aggregated solids were removed. The clarified broth was concentrated to a MW of 100 kDa to 500 kDa. The solution was diafiltered using a CO (molecular weight cut-off) filter. Diafiltration using MgCl2 and sodium phosphate buffers Diafiltration removed residual nucleic acids and proteins.

[0131] Additionally, sodium acetate and sodium acetone with denatured alcohol and / or isopropanol Impurities were removed by reprecipitation of the polysaccharide in phenol and then ethanol. The diafiltered retentate was diluted with sodium acetate in sodium phosphate saline buffer. Thorium and phenol (liquid or solid phenol) were added. The alcohol fractionation of the glycosides was carried out in two stages. In the first stage, the preparation contained a low percentage of alcohol. The ethanol was added to precipitate cell debris and other undesirable impurities, while the crude polysaccharide remained in solution. Impurities were removed by centrifugation followed by a depth filtration step. Additional isopropyl alcohol was then added. The polysaccharides are recovered from solution by adding propanol or denatured alcohol to the batch. The precipitated polysaccharide pellet was collected by centrifugation, ground, and dried to a powder. The cells were stored frozen at -70°C.

[0132] [Example 2] NMR structural analysis of polysaccharides Strategies for determining polysaccharide structures include those described by Abeygunawardana et al. .,Determination of the Chemical Structure e of Complex Polysaccharides by Heteronu clear NMR Spectroscopy in Advances in Bi Physical Chemistry 1993,Vol 3,pages 199 -249, multiple acts substantially as described in JAI Press Inc. The purified polysaccharide was analyzed using standard 1D and 2D NMR techniques. Finally, we investigated the glycosides. 31P NMR was used to characterize the polysaccharide for the presence of phosphate. The body was examined.

[0133] 1 H- 1 H COSY, double quantum filter homonuclear COSY(double quantum m filtered homonuclear COSY) and total correlation spectroscopy (TO The assignment of monosaccharide residues was performed by heteronuclear single quantum coherence spectroscopy (HETS). eronuclear single quantum coherence spec HSQC (High-Speed ​​Quantitative Analysis), and a combination of HSQC-TOCSY, 13 C Chemical shifts were assigned. Multiplicity-edited HSQC was used to distinguish between methylene and methine groups. The inter-residue bonds were determined by a combination of HMBC and NOESY spectroscopy. -Proton and carbon chemical shifts, 3 J H1、H2 and 1 J H1、C1 value of residue The anomeric configuration was determined. 3 J H1、H2 and 1 J H1、C1 From the value, the a of the furanose ring Since it is difficult to assess the anomeric configuration, the ring protons from the anomeric carbon are used in this determination. The long-range coupling was utilized.

[0134] 1D phosphorus NMR spectroscopy showed that the serotype 24F polysaccharide contains phosphorus in its structure. For the prepared (Fig. 5A) and periodate-oxidized (Fig. 5B) polysaccharides 1 H- 31 The phosphorus binding site was assigned by P HMBC spectroscopy.

[0135] Based on the NMR data in Figures 2 to 5, the structure of S. pneumoniae serotype 24F polysaccharide was determined. It is decided as follows: [ka] where n represents the number of repeating units that make up the polysaccharide. See also Figure 1A.

[0136] The sugar residues are rhamnose (Rha), ribose (Rib), glucose (Glc) and It contains arabinitol. One of the glucose residues is N-acetylated. Ribose is It is in the form of furanose.

[0137] The italics (p and f) indicate pyranoses (closed rings of six atoms) and furanoses. It refers to a ring structure (a closed ring consisting of five atoms).

[0138] α and β refer to the configuration of the proton attached to the anomeric carbon of the sugar unit. When labeling carbon atoms, the anomeric carbon is always number 1 (usually 1-6). - means that the proton is in the equatorial position in the 3D structure. This means that the ton is in the axial position.

[0139] The numbers associated with the arrows indicate how the individual sugar units are connected to each other. For example, the name α-Rhap-(1→3)-α-Glcp- was chosen by Rhamnoides. This means that the first carbon of glucose is bonded to the third carbon of glucose (p means that (This also means that it is a pyranose ring).

[0140] Identification of the major activation site of S. pneumoniae serotype 24F polysaccharide The primary activation site of S. pneumoniae serotype 24F is the second carbon of the arabinitol side chain. It was found that.

[0141] The oxidized serotype 24F polysaccharide was lyophilized and then resuspended in deuterium oxide for NMR analysis. Using a cryogenically cooled probe, the 600M NMR experiments were performed at 10 Hz with 16 transient 90 degree pulses and a 10 second delay between pulses. One-dimensional (1D) proton spectra were acquired using a 100 Hz pulse duration (including an acquisition time of 3 seconds). 4 and 16 transients (respectively) in the first dimension, and 512 increments in the second dimension. Gradient COSY and TOCSY data were acquired using the recycle time of 10 seconds ( 5.1 with 1024 transients at 3 s acquisition time and 90 ms mixing time A 1D TOCSY was acquired centered on the triplet at 9 ppm. 64 transients and multiplicity-edited gHSQCAD was obtained with 512 increments.

[0142] The 1D proton spectrum shows a shift in the anomeric region at 5.19 ppm compared to the non-activated polysaccharide. The gCOSY and TOCSY data showed an additional signal (triplet) in the region A cross peak was observed from a signal at 5.19 ppm to a signal at 3.86 ppm. 1D TOCSY with a selective band centered at .19 ppm shows a 3.86 ppm The gHSQCAD data showed only a single correlation with the triplet (Figure 6). 3 ppm (signal at 5.19 ppm) and 70.79 ppm (signal at 3.86 ppm). The carbon chemical shifts at 91.13 ppm were obtained for the anomeric This is slightly upfield from where the signal is typically observed, and is the hydrated aldehyde. The other signal at 70.69 ppm is a methylene. (In a multiplicity-edited gHSQCAD experiment, the methylene carbon was identified as the methine carbon and (Figure 7). The data show that the proton at 5.19 ppm (water The methylene group (protons at 3.86 ppm and The only place this can occur is in the 24F polysaccharide. Based on the structure of the compound, it is at carbon 2 on the arabinitol side chain.

[0143] The formation of aldehydes mainly at the second carbon is due to the preference for the 2,3-carbon by vicinal diols. or at higher carbon numbers by vicinal diols (or aldehydes). A preferred sequence of oxidations is required until the final oxidation at the second carbon of the arabinitol sugar. It was surprising in that it was essential.

[0144] [Example 3] S. pneumoniae to CRM197 using reductive amination in dimethyl sulfoxide Conjugation of E. coli serotype 24F polysaccharide The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and dehydrated by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and then washed with humidifier. The redissolved polysaccharide and CR were then redissolved in dimethyl sulfoxide (DMSO). The M197 solutions were combined and conjugated as follows: Final 0.2 micrograms Prior to filtration, the resulting conjugate was purified by ultrafiltration. Several process parameters within each step, such as temperature and time, are controlled to obtain the desired A conjugate having the desired properties was obtained.

[0145] Polysaccharide size reduction and oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 microns. The size of the polysaccharides was determined by adding acetic acid to 200 mM and incubating at 92°C for 50 minutes. and then neutralized by adding cold potassium phosphate pH 7 buffer to 400 mM. It was reduced by acid hydrolysis.

[0146] The size-reduced polysaccharides were concentrated and purified using a 5 NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered against

[0147] The polysaccharide solution was then adjusted to 22° C. and pH 5 using sodium acetate buffer. Size reduction of polysaccharides due to activation was minimized. 100 mM sodium metaperiodate The activation of polysaccharides was initiated by adding the ethanol solution. The target level of polysaccharide activation was To achieve this, sodium metaperiodate was added per mole of polysaccharide repeat unit. The amount of sodium metaperiodate per unit area was 0.18-0.21 mol (polysaccharide repeat unit). The oxidation reaction was allowed to proceed for 2 hours at 22°C.

[0148] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. The solution was then diluted with water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0149] Conjugation of polysaccharides to CRM197 As previously described (WO2012 / 173876A1), Pseudomonas fluorescens Purified CRM197 obtained by expression in 5 kDa NMWCO tangential flow ultrafiltration was Diafiltration was performed using a filtration membrane against 2 mM phosphate, pH 7.0 buffer. The solution was filtered through a 0.2 micron filter.

[0150] The activated polysaccharide was lyophilized at 2 mg Ps / mL with a sucrose concentration of 10% w / v. CRM197 was formulated for drying. / mL and formulated for lyophilization.

[0151] The formulated Ps and CRM197 solutions were lyophilized separately. The s and CRM197 materials were separately redissolved in equal volumes of DMSO. Sodium was added to a final concentration of 20-50 mM. Blend the above to obtain a polysaccharide concentration of 1.4-1.5g Ps / L (grams of polysaccharide per liter). ) and a mass ratio of polysaccharide to CRM197 of 1.5 was achieved. The ratio of polysaccharide to CRM197 in the conjugate was controlled. Add sodium (1 mole per mole of polysaccharide repeating unit) and allow to conjugate for 2 hours at 22°C. The project progressed.

[0152] Reduction with sodium borohydride Following the conjugation reaction, sodium borohydride (polysaccharide repeat unit 1) was added. The mixture was incubated at 22°C for 1 hour. At approximately 4°C, approximately 0.025 The batch was diluted with 150 mM sodium chloride containing % (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. For some batches, The batch was concentrated and filtered using a 30 kD NMWCO tangential flow ultrafiltration membrane to 150 mM sodium chloride. Diafilter against 25 mM potassium phosphate pH 7 at approximately 4°C. Ta.

[0153] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to 0. In 150 mM sodium chloride pH 7.0 containing .015% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine at 4°C.

[0154] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. with 10 mM histidine in additional 150 mM sodium chloride pH 7.0 containing phosphate 20 Dilute, aliquot and freeze at ≦-60°C.

[0155] Table 1 shows the S. pneumoniae serotype 24F polysaccharide conjugates prepared in DMSO. Indicates the attributes of. [Table 1]

[0156] [Example 4] Formulation of monovalent conjugates Pneumococcal polysaccharide-CRM197 conjugates were prepared as described in Example 3. Based on the batch volume and concentration of the individual bulk polysaccharide concentrations, the target The required amount of bulk conjugate required to achieve the target concentration was calculated. The bulk conjugate of purified type 24F was combined with excipients, sterile filtered, and mixed under A PA was added. 20 mM histidine, 150 mM NaCl, 0.2% (w / v) PS- 20, and S. pneumophila containing 0.250 mg / mL (w / v Al) in the form of APA. The final concentration of the NIE serotype 24F monovalent conjugate vaccine was 4 μg / mL (w / v P nPs).

[0157] [Example 5] Monovalent conjugate immunogenicity study in New Zealand white rabbits Immunogenicity of monovalent conjugates in the New Zealand White rabbit (NZWR) model On days 0 and 14 (alternate sides), each monovalent conjugate vaccine was administered Adult New Zealand White rabbits (NZWR, n = 3 / group) were intramuscularly injected with 0.25 ml of The mice were immunized intramuscularly (IM). 62.5 μg of aluminum phosphate adjuvant was used per immunization. 1 μg of PnPs (S. nuclei conjugated to CRM197) was incubated with 1 μg of 10 ... The subjects were administered a monovalent pneumococcal vaccine at a dose of 100 mg / kg / day (pneumococcal serotype 24F polysaccharide). Before immunization), on day 14 (post-injection 1, PD1), and on day 28 (post-injection 2, PD2). Animals were examined by trained animal care staff at NZW R was monitored at least daily. The vaccine formulation against NZWR appears to be safe and well tolerated. All animal studies were conducted in accordance with the National Institutes of Health's Guide for Care and d Strictly follow the recommendations in the Use of Laboratory Animals. The NZWR experimental protocol was developed by Merck & Co., Inc. (Kenilworth, MA, USA). , NJ) and Covance (Denver, PA) Approved by the Animal Care and Use Committees.

[0158] NZWR sera were assayed using ELISA using each PnPs coating concentration of 1–2 mg / ml. The immunogenicity of IgG was evaluated by testing with the opsonase A assay based on the previously described protocol. Functional antibodies were determined by immunophagocytosis assay (OPA). Aguilar et al., 2017, Vaccine 35:865-72 and Burton et al.,2006,Clin Vaccine Immunol Please see 13(9):1004-9.

[0159] A monovalent pneumococcal conjugate vaccine derived from S. pneumoniae serotype 24F was administered to rabbits. The results showed that the antibodies were immunogenic against each of the bacterial strains (Figure 8) and produced functional antibodies that killed each of the bacterial strains. (Figure 9).

[0160] [Example 6] Monovalent conjugate mouse challenge assay To verify the immunogenicity of S. pneumoniae serotype 24 glycoconjugates, mouse They were tested in a challenge model. Inoculate juvenile mice (6-12 months) with 1 ml of 24F-CRM197 monovalent conjugate vaccine. 8 weeks old) were immunized (n=10 / group). C57BL / 6 mice and Swiss Web ster mice were immunized intraperitoneally (IP) with 0.1 ml injected at a single site. Each mouse was immunized intramuscularly (IM) with 0.05 ml injected at two sites. or 0.4 μg of Pn with 25 μg of aluminum phosphate adjuvant (APA). Ps (24F polysaccharide conjugated to CRM197) monovalent pneumococcal vaccine Mice were examined by trained animal care staff for signs of illness or distress. The vaccine formulation was considered safe and well tolerated in mice. It was.

[0161] On day 52, mice were challenged intratracheally with S. pneumoniae serotype 24F. Log-phase cultures of S. pneumoniae were centrifuged, washed, and suspended in sterile PBS. Mice were anesthetized with isoflurane prior to insertion. In the throat, 10 in 0.1 ml of PBS 6 cfu of S. pneumoniae were placed in the larynx. The tongue was gently pressed against the outside. The mice were then pulled tightly and their nostrils were covered to induce bacterial aspiration. Mice were euthanized if their body weight loss exceeded 20% of their starting body weight. Blood was drawn at 10 and 72 hours to assess bacteremia. Mice were observed at least twice daily by trained animal care staff. Both are based on the National Institutes of Health's Guide for Care and Use of La The experimental procedures for mice were carried out in strict accordance with the recommendations of the International Society for Clinical Animals. Tocol is a product of the Institutional Animal Approved by the Care and Use Committee.

[0162] The 24F-CRM197 monovalent pneumococcal conjugate vaccine is a monovalent pneumococcal vaccine that is designed to protect against S. pneumoniae 24 The results showed that the antibody produced protective effects in all three mouse strains after challenge with F. 0~See FIG. 12.

[0163] [Example 7] Formulation of a pneumococcal conjugate vaccine for rabbit multivalent testing. The pneumococcal polysaccharide-CRM197 conjugate was used to investigate the effect of various conjugate ballasts. Cblend preparations (S. pneumoniae serotypes 16F, 23A, 23B, 24F and 31 A multivalent pneumococcal conjugate vaccine consisting of a polysaccharide derived from Streptococcus pneumoniae was prepared, and the total polysaccharide concentration was 84μg / mL of erythropoietin, 4μg / mL of each serotype, 20mM histidine pH 5.8 and and 150 mM sodium chloride and 0.1% w / v polysorbate-20 (PS-20 ) was formulated into pneumococcal polysaccharide (PnPs) types (S. pneumoniae serotypes 16F, 23 CRM197 protein was individually cloned into each of the 23A, 23B, 24F and 31 derived The conjugates were prepared by conjugating the individual bulk polysaccharide concentrations. Based on the catch volume and concentration, the bulk conjugates required to achieve the target concentration of each serotype were The amount of conjugate required was calculated. Each conjugate was mixed with histidine, sodium chloride and and polysorbate-20 (PS-20) solutions to create the conjugate blend. The formulation vessel containing the conjugate blend was mixed using a magnetic stir bar and sterile filtered. The formulation was then filtered into a separate container. Alternatively, the solution was filled into vials and stored at 2 to 8°C.

[0164] [Example 8] Immunogenicity of a polyvalent pneumococcal conjugate vaccine in New Zealand White rabbits On days 0 and 14 (alternate sides), the 21-valent pneumococcal conjugates described in Example 7 were administered. Adult New Zealand White rabbits (NZWR, n=5) were vaccinated with 0.5 ml of GATE vaccine. Each mouse (1 / group) was immunized intramuscularly (IM) with 2 μg of each conjugate PnPs per immunization. The PCV21 pneumococcal vaccine was administered before the start of the study (pre-immunization) and on the 14th day (post-immunization). Serum was collected on days 1 (post-dose 1, PD1) and 28 (post-dose 2, PD2). NZWR were observed at least daily by trained animal care staff for signs. The vaccine formulation against ZWR was deemed safe and well tolerated. , National Institutes of Health's Guide for Care and Use of Labor The experimental protocol was carried out in strict accordance with the recommendations of the National Institute for Animal Science (NZWR). is an Insulator of Merck & Co., Inc. and Covance (Denver, PA). Titional Animal Care and Use Committee Approved by s.

[0165] Multiplexed electrochemiluminescence Evaluate IgG immunogenicity of NZWR sera using an electron chromatin (ECL) assay Using SULFO-TAG™ labels that emit light upon electrochemical stimulation, soScale Discovery(MesoScale Diagnostics, Using technology developed by NVIDIA, a division of NVIDIA Corporation, LLC, Gaithersburg, MD , Marchese et al.(Optimization and valida tion of a multiplex,electrochemiluminesc ence-based detection assay for the quant itation of immunoglobulin G serotype-spe cific antipneumococcal antibodies in hum an serum.Clin Vaccine Immunol.16(3):387- 96 (2009)) and used for rabbit serum. This assay was developed to test NZWR serum samples using SU as the secondary antibody. LFO-TAG™-labeled anti-rabbit IgG was used. )3Software (UAB Research Foundation, Caro-Ag Uilar et al., 2017, see above; Burton et al., 2006 (see above) from the Bacterial Res. At the piratory pathogen reference laboratory Based on the above protocols available online, multiplexed opso Functional antibodies were determined by molecular phagocytic assays (MOPA).

[0166] S. pneumoniae serotypes 16F, 23A, and 31A in multivalent pneumococcal conjugate vaccines Polysaccharide-protein conjugates prepared from 23B, 24F and 31 were The drug was immunogenic in rabbits at both post-injection 1 (PD1) and post-injection 2 (PD2) These were also found to produce functional antibodies that killed vaccine-type bacterial strains. Immunization with a 2 μg dose of the multivalent pneumococcal conjugate vaccine was performed ( FIG. 14 ). In rabbits treated with immunization, the MOPA of PD1 was significantly higher for the four serotypes compared to pre-immune rabbit sera. The titers were significantly higher in rabbits immunized with a 2 μg dose of PCV21 (Figure 14). showed that the MOPA activity of PD2 was significantly increased for all five serotypes compared to pre-immune rabbit sera. The L value was significantly higher than that of the control group (Figure 14). OG transformed data were analyzed to determine significance.

Claims

1. An activated polysaccharide comprising the structure: 【Chemistry 1】 [In the formula, n represents the number of repeating units].

2. 2. The activated polysaccharide of claim 1, wherein the activated polysaccharide has a molecular weight of from 50 kDa to 1,000 kDa.

3. 2. The activated polysaccharide of claim 1, wherein the activated polysaccharide has a molecular weight of from 100 kDa to 800 kDa.

4. 2. The activated polysaccharide of claim 1, wherein the activated polysaccharide has a molecular weight of from 100 kDa to 300 kDa.

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