Pneumococcal polysaccharides and use thereof in immunogenic polysaccharide-carrier protein conjugates

Abstract

To provide purified capsular polysaccharides from Streptococcus pneumoniae serotypes 23A and 23B, and polysaccharide-protein conjugates having these serotypes.SOLUTION: The present invention provides capsular polysaccharides from Streptococcus pneumoniae serotypes identified using NMR. The present invention further provides polysaccharide-protein conjugates in which capsular polysaccharides from one or more of these serotypes are conjugated to a carrier protein such as CRM197. Polysaccharide-protein conjugates from one or more of these serotypes may be included in multivalent pneumococcal conjugate vaccines having polysaccharides from multiple additional Streptococcus pneumoniae serotypes.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to a method for the treatment of Streptococcus pneumoniae. Purified capsular polysaccharides from Bacillus umoniae serotypes 23A and 23B, and Polysaccharide-protein conjugates having polysaccharides derived from one or more of the serotypes Polysaccharide-protein conjugates derived from one or more of these serotypes are provided. The gate 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 death 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. Some studies have shown that pneumococcal meningitis can cause mortality of up to 8% and 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 proven to be extremely beneficial in preventing pneumococcal disease in people at high risk. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell-independent immunogens that induce weak or no responses in infants. Chemical conjugation of bacterial polysaccharide immunogens to carrier proteins has been shown to enhance immune responses in infants. Diphtheria toxoid (a chemically detoxified version of DT) is used to change the immune response to T-cell dependent responses. DTx and CRM197 have T cell stimulatory epitopes in their amino acid sequences. It has been described as a carrier protein for bacterial polysaccharide immunogens.

[0004] The pneumococcal conjugate vaccine, Prevnar®, was initially used in infants and toddlers. The seven most frequently isolated serotypes (4, 6) causing invasive pneumococcal disease in 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 is limited, and in the United States, certain serotypes (e.g., 1 There is some evidence that new species (e.g., 9A) are emerging. 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 1, 3, 4, 5, 6A, 6B, 7F, and 9V 13-valent pneumococcal polysaccharide-protein complex containing 14, 18C, 19A, 19F, and 23F Conjugate vaccines are described, for example, in U.S. Patent Application Publication No. 2006 / 022838 0A1, Prymula et al., 2006, Lancet 367:740-4 8, and 48 in Washington, D.C., 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 Mi Crobiology Reviews 28:871-899. Lear Patent No. IT1418572B1 describes serotype 23A, but the structure is not provided. It has not been done.

[0007] Current multivalent pneumococcal conjugate vaccines are based on the serotypes present in the vaccine, e.g. It is effective in reducing the incidence of 23F-associated pneumococcal disease. The prevalence of pneumococci expressing serotypes not present in the current study is increasing. A pneumococcal conjugate vaccine containing Streptococcus pneumoniae provided cross-protection against serotypes 23A and 23B. Therefore, emerging pneumococcal serogroups should be considered for inclusion in future vaccines. The types need to be identified and characterized. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent Application Publication No. 2006 / 0228380A1 [Patent Document 2] Italian Patent No. IT1418572B1 [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

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[0010] The present invention provides purified Streptococcus pneumoniae serotypes 23A and 23B derived The present invention provides capsular polysaccharides containing these serotypes, as well as polysaccharide-protein conjugates containing these serotypes. The present invention is based in part on the structural identification of capsular polysaccharides from these serotypes. [Means for solving the problem]

[0011] Thus, in one embodiment, the present invention provides a polysaccharide having any of the following repeating units: Offering the body: [ka] The polysaccharide from Streptococcus pneumoniae serotype 23A is represented by And, [ka] In the formula, n represents the number of repeating units.

[0012] The polysaccharide from Streptococcus pneumoniae serotype 23B may be represented by And, [ka] In the formula, n represents the number of repeating units.

[0013] In certain embodiments, the polysaccharide has between 10 and 5,000 repeating units. In this case, polysaccharides have 50 to 3,000, 100 to 2,500, or 100 to 2,000 repeating units. It has a repeat unit.

[0014] In certain embodiments, the polysaccharide has a molecular weight of between 50 kDa and 4,000 kDa. In certain embodiments, the polysaccharide is between 80 kDa and 2,000 kDa or between 100 kDa and 1,500 kDa. It has a molecular weight of 0 kDa.

[0015] The present invention further provides an activated polysaccharide produced from any of the above embodiments, wherein In this procedure, polysaccharides are activated by chemical reagents and attached to linker or carrier proteins. In certain embodiments, the S. pneumoniae Activation of serotype polysaccharide 23A occurs with α-Rhap or β-Glcp. In morphology, activation of S. pneumoniae serotype polysaccharide 23B is mediated by β-Glcp or β-R In one aspect of this embodiment, the activity of S. pneumoniae serotype polysaccharide 23B Activation occurs in excess of 90%, 95%, or 99% of β-Rhap. In certain aspects of this embodiment, the polysaccharide is activated by periodate. Activation of type 23A polysaccharides occurs at the second or third carbon position of α-Rhap or β-Glcp. Alternatively, activation of serotype 23B polysaccharide occurs via β-Glcp or β-Rhap. In a subembodiment of this embodiment, the serotype 23B polysaccharide is Periodate activation of the body occurs in >90%, 95%, or 99% of cases with β-Rhap .

[0016] The present invention further provides a method for preparing a polysaccharide or activated polysaccharide as provided above, which is attached to a carrier protein. In certain embodiments, polysaccharide-protein conjugates are 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 derived from Bacillus subtilis (L. ginosa). 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. They are prepared using selective amination chemistry.

[0018] In one embodiment, the present invention provides a method for the detection of Streptococcus pneumoniae serotypes 23A and 23B. unconjugated polysaccharides or polysaccharide-protein conjugates derived from one or more of the 3B Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, and 6 B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 2 1, 22A, 22F, 23F, 24B, 24F, 27, 28A, 31, 33F, 34, 3 unconjugated polysaccharides derived from one or more of 5A, 35B, 35F and 38 or a polysaccharide-protein conjugate. In a subembodiment, the multivalent immunogenic composition comprises an unconjugated polysaccharide or a polysaccharide-capsule. In one subembodiment, the polynucleotide comprises a polynucleotide conjugate, but not both. The covalent immunogenic composition may comprise an unconjugated polysaccharide or a polysaccharide-carrier protein conjugate. In certain subembodiments, the multivalent immunogenic compositions of the invention comprise a mixture of at least Large 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, 3 3, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or There are 90 serotypes. [Brief explanation of the drawings]

[0019] [Figure 1A] A pictorial representation of the repeating unit structure of S. pneumoniae serotype 23A (A) and 23B (B) polysaccharides is shown. [Figure 1B] A pictorial representation of the repeating unit structure of S. pneumoniae serotype 23A (A) and 23B (B) polysaccharides is shown. Activation sites available for periodate are indicated by arrows. In activated polysaccharides, not every activation site in the repeating unit is activated. This reflects the possibility of activation of serotype 23B polysaccharide at the second or third carbon position of β-Rhap. [Figure 2A] 600 MHz 1D 1H NMR spectra of capsular polysaccharides from S. pneumoniae serotypes 23A (A) and 23B (B) in deuterium oxide (DO) at 50° C. [Figure 2B]Figure 1 shows 600 MHz 1D H NMR spectra of capsular polysaccharides from S. pneumoniae serotypes 23A (A) and 23B (B) in deuterium oxide (DO) at 50°C. Signals resulting 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. [Figure 3A] 1 shows the one-dimensional (1D) 1H NMR identity regions used for serotype identification of S. pneumoniae serotypes 23A (A) and 23B (B). [Figure 3B] The one-dimensional (1D) H NMR identity regions used for serotype identification of S. pneumoniae serotypes 23A (A) and 23B (B) are shown. The signal positions of each anomeric proton of the repeating unit from each monosaccharide residue are indicated. [Figure 4A] Partial two-dimensional (2D) 1H- 13C multiple bond correlation NMR spectra of S. pneumoniae serotypes 23A (A) and 23B (B) are shown, establishing covalent bonds between the sugar residues of the repeating structures. [Figure 4B] Partial two-dimensional (2D) H-C multiple bond correlation NMR spectra of S. pneumoniae serotypes 23A (A) and 23B (B) are shown, establishing covalent bonds between the repeating sugar residues. The correlations establishing the glycosidic bonds are indicated in the figure. [Figure 5A] Establishment of phosphodiester bonds within the capsular polysaccharide repeat unit of S. pneumoniae serotypes 23A (A) and 23B (B). [Figure 5B] Establishment of phosphodiester bonds within the capsular polysaccharide repeat unit of S. pneumoniae serotypes 23A (A) and 23B (B) is shown. [Figure 6] 600 MHz 1D H NMR spectrum of oxidized and TSC-derivatized capsular polysaccharide from S. pneumoniae serotype 23B in deuterium oxide (DO) at 25° C. The inset is a magnification of the imine signal formed by derivatization with thiosemicarbazide. [Figure 7] 2D TOCSY of the spectrum of oxidized and TSC-derivatized capsular polysaccharide from S. pneumoniae serotype 23B. The peaks at 7.36–7.40 ppm correlated with the rhamnose CH3 peak (approximately 1.32 ppm). The inset shows the structure of capsular polysaccharide from S. pneumoniae serotype 23B, with the periodate activation site indicated by an arrow. [Figure 8A] 2D gCOSY (A) and NOESY (B) spectra of oxidized and TSC-derivatized capsular polysaccharide of S. pneumoniae serotype 23B. [Figure 8B] 2D gCOSY (A) and NOESY (B) spectra of oxidized and TSC-derivatized capsular polysaccharide of S. pneumoniae serotype 23B. [Figure 9] ELISA IgG antibody titers (post-dose 2) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Error bars represent the geometric mean + 95% confidence interval. [Figure 10] Serotype-specific OPA titers (post-dose 2) in rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Error bars represent the geometric mean + 95% confidence interval. [Figure 11A] ELISA PnPs23A IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23A OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). [Figure 11B]ELISA PnPs23A IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23A OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Error bars represent the geometric mean + 95% confidence interval. [Figure 12A] ELISA PnPs23B IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23B OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). [Figure 12B] ELISA PnPs23B IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23B OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Error bars represent the geometric mean + 95% confidence interval. [Figure 13A] ELISA PnPs 23F IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23F OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). [Figure 13B]ELISA PnPs 23F IgG antibody titers (A) and S. pneumoniae polysaccharide serotype 23F OPA titers (pre-immunization, post-dose 1 (PD1), and post-dose 2 (PD2)) (B) of rabbits immunized with S. pneumoniae monovalent polysaccharide serotypes 23A, 23B, or 23F conjugated to CRM197 and formulated in aluminum phosphate adjuvant (APA). Error bars represent the geometric mean + 95% confidence interval. [Figure 14] Serotype-specific (S. pneumoniae serotypes 16F, 23A, 23B, 24F, 31) pre-immunization, PD1, and PD2 geometric mean antibody titers are shown for rabbits immunized with a multivalent pneumococcal conjugate vaccine (2 µg / PnPs). Error bars represent two standard errors of the geometric mean titer for each serotype (X-axis). [Figure 15] Serotype-specific (S. pneumoniae serotypes 16F, 23A, 23B, 24F, and 31) pre-immunization, PD1, and PD2 OPA dilution titers are shown for rabbits immunized with a multivalent pneumococcal conjugate vaccine (2 μg / PnPs). Symbols indicate individual titers, and error bars represent the 95% confidence interval (CI) of the geometric mean titer (GMT). *p<0.05, **p<0.01, ***p<0.001; ns=not significant. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based in part on the identification of novel pneumococcal polysaccharide structures by NMR techniques. The structures provided herein are the most representative of these S. pneumoniae serotypes 23A and 23B. It is believed to be the first identification or the first correct identification.

[0021] S. pneumoniae serotype 23A and 23B polysaccharides produced by their respective strains The produced (and purified) polysaccharides were used to characterize individual Ps-C RM197 conjugate was produced. S. pneumoniae serotypes 23A and 23B were It has a unique polysaccharide structure, which allows the conjugate production process to take place. The conjugate was demonstrated to be immunogenic in animal studies.

[0022] The term "polysaccharides" (Ps) as used herein includes, but is not limited to, "sugars", "Oligosaccharides," "polysaccharides," "liposaccharides," "lipooli Lipopolysaccharide (LOS), Lipopolysaccharide (LPS), Glycosylate, Glycoconjugate immunological and cytochemical studies, including "antibody gates," "derivatized or activated polysaccharides or oligosaccharides," etc. It may contain any antigenic sugar element (or antigenic unit) commonly used in the field of bacterial vaccines. Unless otherwise specified, the names of polysaccharides used herein refer to 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 immunogenic composition that is administered to a host, such as a mammal, by injecting the immunogenic composition into the body fluids of the host. 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 containing an antigen. An immunogenic composition is an antigen associated with an MHC molecule on the cell surface. Furthermore, the presentation of antigens can help sensitize the host. can be produced to provide future protection to the immunized host. The composition can protect the host from infection by bacteria, reduce the severity of the infection, or The immunogenic composition may also be able to: It can be used to generate polyclonal or monoclonal antibodies, and polyclonal Alternatively, monoclonal antibodies can be used to confer passive immunity to a subject. The immunogenic compositions are used in animal efficacy models or opsonophagocytic injury assays. Functional antibodies may be generated as measured by killing of bacteria via the antibody.

[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 contain proteins, nucleic acids, and nonspecific endogenous polysaccharides (C polysaccharides). The isolated polysaccharides are those that are partially removed from the soluble fraction of the soluble polysaccharides. The isolated polysaccharides contain less than % of 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 polysaccharides refer 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 Mw is expressed in kDa. The Mw is the amount of large molecules a polymer sample contains more than small molecules. The total mass of Mw is calculated 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 NMR or proton NMR. Reflects variability.

[0028] As used herein, the term "molar ratio" is typically expressed in tenths or hundredths. 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 admixtures). (The term "composed of" applies to compounds of the present invention.) The term "consisting of" when used in conjunction with conjugate mixtures refers to those specific S. pneumoniae polysaccharide-protein conjugates and other S. pneumoniae from different serotypes . Refers to a mixture that does not have 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

[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 in which all available activation sites have been 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 include the lower and upper limits recited. Including limitations.

[0035] Two additional S. pneumoniae serogroup 23 strains for which no information on structure or composition is available Additional members have been identified. The serotype 23B polysaccharide has the same backbone as the serotype 23F polysaccharide. but lacks the pendant α-Rhap. The type 23A polysaccharide has a short backbone and long side chains.

[0036] By identifying the structures of these serotypes, it is possible to identify unconjugated polysaccharides or polysaccharide- It will be possible to incorporate them into pneumococcal vaccines as protein conjugates. Conjugate vaccines containing streptococcal and pneumococcal Ps are well known in the art. For example, U.S. Patent No. 6,248,570, U.S. Patent No. 5,866,135, and and U.S. Patent No. 5,773,007.

[0037] capsular polysaccharide Pods from Streptococcus pneumoniae from the serotype(s) of the present invention Membrane polysaccharides can be prepared by standard techniques known to those skilled in the art. For example, can be isolated from bacteria and prepared by known methods (see, for example, European Patent No. EP499999). 7524 and European Patent No. EP497525), preferably a homogenizer To some extent, size can be achieved by microfluidization or chemical hydrolysis. In one embodiment, S. pneumoniae strains corresponding to each polysaccharide serotype are grown on soybean plants. The cells are grown in a medium containing 100% ethanol, then subjected to standard steps including centrifugation, precipitation, and ultrafiltration. Thus, individual polysaccharides are purified. See, for example, U.S. Patent Application Publication No. 2008 / 028683 See U.S. Patent No. 5,847,112 and U.S. Patent No. 5,847,112. Polysaccharides can be sized to form viscosity-enhancing agents. reducing the degree of cleavage and / or improving the subsequent filterability of the conjugated product Chemical hydrolysis can be carried out using acetic acid. Mechanical sizing can be carried out using high pressure hoses. This may be done using homogenizing 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 radioisotope detector (RI). In other such embodiments, the polysaccharide may be between 10 kDa and 4,000 kDa. a, 50kDa~4,000kDa, 50kDa~3,000kDa, 50kDa~2, 000kDa, 50kDa~1,500kDa, 50kDa~1,000kDa, 50kDa Da~750kDa, 50kDa~500kDa, 80kDa~2000kDa, 100 kDa~4,000kDa, 100kDa~3,000kDa, 100kDa~2,00 0kDa, 100kDa~1,500kDa, 100kDa~1,000kDa, 100 kDa~750kDa, 100kDa~500kDa, 100~400kDa, 200kDa Da~4,000kDa, 200kDa~3,000kDa, 200kDa~2,000 kDa, 200kDa to 1,500kDa, 200kDa to 1,000kDa or 20 In a specific embodiment, the antibody from serotype 23A has an average molecular weight of 0 kDa to 500 kDa. The polysaccharide has an average molecular weight of 75 kDa to 200 kDa. The polysaccharide derived from the type 23B has an average molecular weight of 150 kDa to 250 kDa.

[0039] In certain embodiments, the S. pneumoniae serotype 23A or 23B polysaccharide is 10 to 5 In certain embodiments, the polysaccharide has 50 to 3,000, 10 In a specific embodiment, the serotype 23A is 0 to 2,500 or 100 to 2,000. In certain embodiments, the polysaccharide derived from S. nucleophila has 97 to 260 repeating units. The polysaccharide derived from H. monnier serotype 23B has 195 to 324 repeating units.

[0040] Carrier proteins The 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. If multiple serotypes are used in a multivalent composition, they may be used with the same carrier protein or different serotypes. Serotypes may be prepared using carrier proteins of different capsular polysaccharides of the same serotype. 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). The protein is 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. Corynebacterium diphtheriae (Co. From a culture of Rynebacterium diphtheria strain C7(β197) In another embodiment, CRM197 is isolated from the ribozyme described in U.S. Pat. No. 5,614,382. CRM197 is typically 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). Pseudomonas fluorescens was used It is prepared in the US.

[0042] Other suitable carrier proteins include additional inactivated bacterial toxins, such as DT, Diphtheria Tetanus toxoid fragment B (DTFB), TT (tetanus toxoid), or TT fragment Fragment C, pertussis toxoid, cholera toxoid (see, e.g., International Patent Application Publication No. W O2004 / 083251), E. coli LT (heat-labile enterotoxin syn), E. coli ST (heat-stable enterotoxin), and Pseudomonas er Also included are bacterial outer membrane proteins, e.g., outer membrane complexes. 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, pneumococcal pneumolysin (Kuo et al., 199 5, Infect Immun 63;2706-13), e.g., non-toxic in some way 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- Keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and Purified protein derivative of tuberculin (PPD), PorB (N. meningitidis (N. meningitidis), PD (Haemophilus influenzae Protein D; see, for example, European Patent No. EP 0 594 610 B), or an immunoglobulin thereof. Epidemiologically Functional Equivalents, Synthetic Peptides (European Patent No. EP0378881 and European Patent No. EP0427347), heat shock proteins (International Patent Application Publication No. WO93 / 17712 and International Patent Application Publication No. WO94 / 03208), pertussis proteins Quality (International Patent Application Publication No. WO98 / 58668 and European Patent No. EP0471177 ), cytokines, lymphokines, growth factors or hormones (see International Patent Application Publication No. (See WO91 / 01146), multiple human CD4 + Artificial proteins containing T cell epitopes (Falugi et al., 2001, E ur J Immunol 31:3816-3824), e.g., N19 protein Quality (Baraldoi et al., 2004, Infect Immun 72:4 884-7), iron uptake proteins (International Patent Application Publication No. WO01 / 72337 C. difficile toxin A or B (see International Patent Application No. See Publication No. WO00 / 61761), and flagellin (Ben-Yedidia et al., 1998, Immunol Lett 64:9) also It can be used as a rear protein.

[0043] As carrier proteins, other DT variants, such as 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 ls and other mutations described by Youle; Glu-148 to Asp, Gln or Ser and / or Ala158 to Gly deletion or mutation, and U.S. Pat. No. 4,709,017 or other mutations disclosed in U.S. Pat. No. 4,950,740 at least one or more residues Lys516, Lys526, Phe530 and / or Mutation of Lys534, and U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,000. 5,673; or other mutations disclosed in U.S. Pat. No. 5,843,711. You can also use fragments.

[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 A protein that is reactogenic and available in sufficient quantity and purity. Proteins may also be used to enhance the immunogenicity of antigens, such as S. pneumoniae polysaccharides. The carrier protein is conjugated or conjugated to a standard conjugation agent. In one embodiment, the first carrier protein is conjugated to the Each ungated capsular polysaccharide is 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 capsular polysaccharide molecule is conjugated to two or more carrier proteins (each In such embodiments, the same serotype Each capsular polysaccharide of is typically conjugated to the same carrier protein.

[0045] Conjugation Prior to conjugation, purified polysaccharides were prepared by reacting the sugars with carrier proteins. The polysaccharides used herein 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. It refers to a polysaccharide that has been chemically modified as described below to enable its action. The purified polysaccharide may be conjugated to a linker. When the capsular polysaccharides are conjugated to the carrier protein, each polysaccharide is conjugated to a glycoconjugate. The polysaccharide conjugates are prepared by known coupling techniques. It is possible.

[0046] In certain embodiments, activation of S. pneumoniae serotype 23A polysaccharide is achieved by α-Rhap or β-Glcp. In certain embodiments, S. pneumoniae serotype 23B polysaccharide. Activation of the catalyzer occurs via β-Glcp or β-Rhap. Activation of serotype 23B polysaccharide was 90%, 95%, 99%, or 100% with β-Rhap. Surprisingly, regardless of the availability of a suitable activation site on β-Glcp, In certain embodiments, the polysaccharide is periodate. In a particular aspect of this embodiment, the activation of serotype 23A polysaccharide is occurs at the second or third carbon position of α-Rhap or β-Glcp, or in serum Activation of type 23B polysaccharides occurs at the second or third carbon position of β-Glcp or β-Rhap. In one subembodiment of this embodiment, the second or third carbon position of β-Rhap The degree of activation of serotype 23B polysaccharide in β-Glcp was determined by the presence of either the second or third carbon position. The degree of activation in β-Rhap is at least 60%, 7 In another subembodiment of this embodiment, the serotype 23B polysaccharide Periodate activation in the body is greater than 90%, 95%, 99% or 100% in β-Rhap. This happens.

[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 a polysaccharide. 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 is carried out by nucleophilic acyl substitution to one of the ester groups of the polysaccharide. This reaction is allowed to occur by reacting with primary amine groups. The polysaccharide is bonded to the linker via a polysaccharide-linker intermediate. The anchor contains a first ester group on the carrier molecule to react with the primary amine groups in the polysaccharide. and a second ester group for reaction with a primary amine group in A typical linker is adipic acid N-hydroxysuccinimide diester (SI DEA).

[0049] In certain embodiments, the coupling is indirect, i.e., coupling to a linker. This can also be done 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; and (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. groups, thereby allowing any of the primary amine groups to be converted to the carboxylate of the polysaccharide by reductive amination. The reaction with the carboxyl group in the polysaccharide allows step (a1) to occur. A primary amine group is used that reacts with the carbonyl group of a hydrazide or hydroxyl Amino groups are suitable. Typically, the additional linker has the same primary amine group at both ends. This reaction results in a polysaccharide bond that is attached to an additional linker via a C-N bond. A linker-addition intermediate is obtained.

[0050] In certain embodiments, different groups of the polysaccharide, particularly the carboxyl groups, are used to provide additional phosphorus. Polysaccharides can be attached to the car. This coupling is done 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 attached to both ends of the It has a primary amine group at the base, so that any of the primary amine groups can be activated by EDAC activation. and reacting with the carboxyl groups of the polysaccharide to allow step (a1) to occur. The primary amine groups are used to react with the EDAC-activated carboxyl groups in the polysaccharide. A hydrazide group is suitable. Typically, the additional linker has the same primary amine at both ends. This reaction connects the polysaccharide to the additional linker via an amide bond. This gives a polysaccharide-additional linker intermediate.

[0051] In one embodiment, chemical activation of the polysaccharide followed by reductive amination of the 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, U.S. Patent Application Publication No. 2 006 / 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 in the presence of an oxidant. with any oxidizing agent that is a primary hydroxyl group to an aldehyde, such as TEMPO. reaction (WO2104 / 097099), or periodate (sodium periodate, Two vicinal compounds for aldehydes, such as potassium iodate or periodate This reaction may involve activation of pneumococcal polysaccharides by reaction of the hydroxyl groups of the polysaccharides. Random oxidation of primary hydroxyl groups of carbohydrates with the formation of reactive aldehyde groups or This results in random oxidative cleavage of vicinal hydroxyl groups.

[0052] In this embodiment, coupling to the carrier protein is via lysyl groups on the protein. For example, conjugation can be achieved by reductive amination via direct amination of The mixture of activated polysaccharide and carrier protein is treated with cyanoborohydride in the presence of nickel. The conjugation reaction is carried out by reacting with a reducing agent such as sodium. , in aqueous solution or in the presence of dimethyl sulfoxide (DMSO). U.S. Patent Application Publication No. US2015 / 0231270 and U.S. Patent Application Publication No. US2 See 011 / 0195086 and 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 oxidation process involves the reduction of a base to form a stable amine conjugate bond. Optionally, the polysaccharides may be reduced in size by mechanical methods (e.g., homogenization) or chemical methods. Hydrolysis may also 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 Both periodate and periodic acid are included; 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 prepared 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, periodate.

[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 the primary hydroxyl, such as and nitroxide radical compounds (see, for example, International Patent Application Publication No. WO2004 / 020944). In the above reaction, the actual oxidant is In one embodiment, the stable nitroxy or nitroxy group is an N-oxoammonium salt. The oxide radical compounds are piperidine-N-oxy or pyrrolidine-N-oxy compounds. In one embodiment, the stable nitroxyl or nitroxide radical compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROX 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 methyl-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-pyridinone as a co-oxidant. Peridinyloxy (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: a) In an aqueous solvent, a sugar is reacted with 2,2,6,6-tetramethyl-1-piperidinyloxy( Activated sugars are reacted with TEMPO and N-chlorosuccinimide (NCS) to form and b) attaching the activated sugar to a carrier protein containing one or more amine groups. (hereinafter referred to as "TE This is called "MPO / NCS reductive amination."

[0056] Optionally, the oxidation reaction may be quenched by adding a quenching agent. Contains vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfur Bisulfite, Dithionite, Metabisulfite, Thiosulfate, Phosphate Phosphite, hypophosphite or phosphorous acid (glycerol, ethylene glycol, Propane-1,2-diol, butane-1,2-diol or butane-2,3-diol The hydroxybenzoates may be selected from the group consisting of hydroxybenzoates, ...

[0057] The second step in the conjugation process for reductive amination is to generate a stable conjugate. Activated polysaccharides using reducing agents to form gate bonds (also known as reductive amination) The reduction of the imine (Schiff base) bond between the carboxyl group and the carrier protein is the preferred reducing agent. cyanoborohydride (e.g., sodium cyanoborohydride) or sodium 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) solvent. If dried, DMSO or DMF solvents are used to prepare activated polysaccharides and carriers. 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 are preferably 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 the reaction of Brønsted or Lewis acids with trimethylsilyl groups. Sodium triacetoxyborohydride or sodium borohydride or borohydride zinc), pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethicone Benzylamine-borane, 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 comprise a glycoconjugate having a molecular weight of 10 kDa to 10,000 kDa. In other such embodiments, the polysaccharide comprises a polysaccharide having a molecular weight of 250 kDa. In other such embodiments, the polysaccharide has a molecular weight of from 0.1 kDa to 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 to 700kDa, 400kDa to 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 facilitate the removal of free cyanide. Transition metals are known to form stable complexes with cyanides, and cyanohydrides are Reductive methylation of protein amino groups with formaldehyde using 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 reduces the remaining inhibitory activity. By forming a complex with an anion, the consumption of the protein during conjugation is increased. This increases the number of conjugates present, leading to 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, activated sugars can be attached to carrier proteins either 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 carrier protein. proteins (e.g., using GMBS) or haloacetylated carrier proteins (e.g., Iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-succinimide (using SIAB, SIA, or SBAP) and The compound can be bound to the carrier via a thioether bond obtained after the reaction. Alternatively, the cyanate ester (which may be made by CDAP chemistry) may be substituted with hexanediamine. Amino-derivatized sugars are conjugated with carbodiimides or adipic acid dihydrazide (ADH), and Imide (e.g., EDAC or EDC) chemistry is used to immobilize the cations on the protein carrier. Such conjugates are conjugated to carrier proteins via carboxyl groups. The patent application is filed under International Patent Application Publication No. WO93 / 15760, International Patent Application Publication No. WO95 / 08348 and International Patent Application Publication No. WO96 / 29094 and Chu et al. l., 1983, Infect. Immunity 40:245-256 There are.

[0064] Other suitable techniques include carbodiimides, hydrazides, active esters, norbornenes, 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 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), 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, optional Optionally, protection / deprotection of primary hydroxyl groups, reaction of primary hydroxyl groups with CDI 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 involved.

[0065] Conjugation (reduction reaction and optionally capping or quenching reaction) The glycoconjugates are then purified (polysaccharide-protein complexes) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration, and Diafiltration / Precipitation, 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 conjugate can be purified by diafiltration or ion exchange chromatography or Purified by size exclusion chromatography.

[0066] One method of characterizing the glycoconjugates of the present 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 available. This can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation of the carrier protein used to create the conjugate material In a preferred embodiment, the number of lysine residues recovered is reduced compared to the starting material. The degree of conjugation of the glycoconjugates of the present invention may be 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 0 to 12. In one embodiment, the conjugation of the glycoconjugate of the present invention 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 , about 13, about 14, or about 15. In a preferred embodiment, the glycoconjugates of the present invention The degree of conjugation of the gate is 4 to 7. In some such embodiments, The carrier protein is CRM197.

[0067] The glycoconjugates of the present invention also have a sugar to carrier protein ratio (wt / wt) of 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, approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9 or In another embodiment, the sugar to carrier protein ratio (w / w) is: 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 carrier protein to the carboxyl group is 0.9 to 1.1. In this example, the carrier protein is CRM197. The immunogenic composition may comprise 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., , non-covalently bound, adsorbed or entrapped in glycoconjugates or glycoconjugates (and thus captured).

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

[0069] Multivalent polysaccharide-protein conjugate vaccines In certain embodiments of the invention, the multivalent polysaccharide vaccine provides a multivalent pneumococcal vaccine. To achieve this, free polysaccharides, components of polysaccharide-protein conjugates, or combinations thereof may be used. Streptococcus pneumoniae serotypes 23A and 23B, or one or more of these and unconjugated polysaccharides or polysaccharide-protein conjugates derived from S. Hemolytic anemia serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8 , 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17 F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23F, 24B, Of 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 and capsular polysaccharides derived from one or more of the following. In certain embodiments of the invention, the immunogenic composition The entities are individually conjugated to one or more carrier proteins. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 S. New Comprising, consisting essentially of, or consisting of capsular polysaccharide from a Monnier serotype. Preferably, saccharides from a particular serotype are conjugated to multiple carrier proteins. It has not been tested.

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

[0071] Cross-protection may be observed for certain serotypes within S. pneumoniae serogroup 23. If so, polysaccharides from a particular serotype may induce a protective immune response against another serotype. Typically, different serotypes are within the same serogroup, but in some cases, one Serotypes may also provide protection against serotypes from different serogroups. No cross-protection has been observed between polysaccharides from these serotypes and those from the same serogroup. Examples include polysaccharides from S. pneumoniae serotypes 23A and 23F that inhibit S. pneumoniae. -Monnier serotype 23B (while providing weak cross-protection against each other) Therefore, the results unexpectedly demonstrate that serotypes 23A, 23B and Multivalent compositions containing polysaccharide serotypes 23A or 23F to provide protection against 23A and 23F The product must contain serotype 23B polysaccharide. Thus, in certain embodiments of the present invention In another embodiment of the invention, the multivalent composition comprises polysaccharides from serotypes 23A and 23B. In the present invention, the polyvalent composition comprises polysaccharides from serotypes 23F and 23B.

[0072] Pharmaceutical / vaccine compositions The present invention further provides the above polysaccharides together with a pharmaceutically acceptable carrier and an adjuvant. comprising, consisting essentially of, or a combination of S. pneumoniae serotypes The present invention provides compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising:

[0073] Formulation of the polysaccharide-protein conjugates of the present invention is art-recognized. For example, individual pneumococcal conjugates can be prepared using methods such as preparing compositions. A physiologically acceptable vehicle may be formulated to achieve this. Examples of such vehicles include: , including but not limited to, water, buffered saline, polyols (e.g., glycerol, protease inhibitors, etc.) pyrene glycol, liquid polyethylene glycol) and dextrose solution.

[0074] In a preferred embodiment, the vaccine composition is an L-histidine buffer containing sodium chloride. It is formulated into a liquid.

[0075] As defined herein, an "adjuvant" is an agent that aids in the immunogenic administration of the immunogenic compositions of the present invention. Immune adjuvants are substances that help increase the virulence of a vaccine. It is immunogenic, e.g., it does not induce any antibody titers, induces weak antibody titers, or is highly immunogenic. Enhances immune responses to antigens that induce cystic immune responses and increases antibody titers against antigens. and / or reduce the dose of antigen effective to achieve an immune response in an individual. Therefore, adjuvants are often administered to enhance the immune response, and are well known to those skilled in the art. Suitable adjuvants to enhance the effectiveness of the composition include, but are not limited to: Includes: (1) Aluminum salts (alum), such as aluminum hydroxide and 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 steroids) Microfluidizer (Microfluidics, Newton, MA) and other microphones 5% squalene, 0.5% T formulated into submicron particles using a Lofluidizer containing Ween 80 and 0.5% Span 85 (containing various amounts of MTP-PE) (b) MF59 (International Patent Application Publication No. WO90 / 14837), (c) It is either microfluidized into a co-emulsion or used to produce a large particle size emulsion. 10% squalene, 0.4% Tween 80, 5% pluronic acid, vortexed for 2 min. SAF containing the block polymer L121 and thr-MDP, (c) 2% squara 0.2% Tween 80, and the 3-aminopropyl methylcellulose described in U.S. Pat. No. 4,912,094. -O-Deacylated Monophosphoryl Lipid A (MPL™), Trehalose Dimycolate Detox (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox ( Ribi™ adjuvants containing one or more bacterial cell wall components from the group consisting of Bunt system (RAS), (Corixa, Hamilton, MT); (d) Montan ide ISA; (3) Quil A or STIMULON™ QS-21 (Antigenic saponin adjuvants such as saponin adjuvants (e.g., U.S. Pat. No. 6,449,293, Framingham, MA) See US Pat. No. 5,057,540) or ISCOMs (cholesterol, saponin , immune stimulating complexes formed by the combination of phospholipids and amphipathic proteins) and Iscomatrix® (which has essentially the same structure as ISCOM but is tamper-resistant) particles produced therefrom, such as (containing no proteins); (4) Available from Corixa and described in U.S. Patent No. 6,113,918 Aminoalkyl glucosamine phosphate compounds (AGP) or their derivatives Bacterial lipopolysaccharides, such as lipopolysaccharides or analogs, and synthetic lipid A analogs; one such AGP is an aqueous form Also known as 529 (previously known as 2-[(R)-3-tetradecanoyloxytetradecanoic acid, previously known as RC529] 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetramethylamino]ethyl tetradecanoyloxytetradecanoyl]-2-[(R)-3- ... 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, e.g., the trimer of complement component C3d.

[0076] 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) It is an oil-in-water emulsion that also contains S21.

[0077] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonine. N-acetyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-aminoglutamine Lanin-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl) methylparaben-2-yl methylparaben (MTP-PE) and the like.

[0078] In certain embodiments, the adjuvant is an aluminum salt. The vaccine may be an alum-precipitated or alum-adsorbed vaccine. Adjuvants for the treatment of rhodium salts 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: 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 oxyphosphate (aluminum phosphate adjuvant (APA)), amorphous aluminum aluminum trihydrate or aluminum trihydroxy.

[0079] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is also known as aluminum chloride. Aluminum hydroxyphosphate is blended with sodium phosphate in a 1:1 volume ratio. After the blending process, a high shear mixer is used to The material is then reduced in size to achieve a monodisperse particle size distribution. The product is diafiltered and steam sterilized.

[0080] In certain embodiments, commercially available Al(OH)3 (e.g., Denmark / Accura te Chemical and Scientific Co.,Westbury, Protein using Alhydrogel® or Superfos® (NY) In another embodiment, protein adsorption occurs at 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 adsorb onto the positively charged aluminum ions over a period of 2-3 weeks. and establish a reservoir of released Ag, inhibiting nonspecific activation of macrophages and complement activity. may be involved in the oxidization and / or stimulate innate immune mechanisms (possibly through stimulation of uric acid) For example, Lambrecht et al., 2009, Curr Opin See Immunol 21:23.

[0081] Monovalent bulk aqueous conjugates are typically blended together and diluted. Once dissolved, the batch is sterile filtered. Aluminium 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.

[0082] 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.

[0083] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "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 See ne 21:4297. In another embodiment, art-recognized uses of the term Any other definition given is intended. A CpG-containing oligonucleotide includes any combination of Modified oligonucleotides using synthetic internucleoside linkages, modified bases and / or modified sugars Includes do.

[0084] The use of CpG oligonucleotides is well known in the art, e.g., 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 ier Syst.23:89-110.

[0085] Administration / Dosage The compositions and formulations of the present invention are suitable for administering vaccines via systemic or mucosal routes. for use in preventing or treating infectious diseases, such as pneumococcal infections, in susceptible humans. In one embodiment, the present invention provides an S. pneumoniae capsular polysaccharide conjugate. A method for eliciting an immune response to a compound 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 infection. A method of vaccinating humans against infection, comprising administering to a subject the immunological effect of an immunogenic composition of the present invention. administering an effective amount to a human.

[0086] The optimal amounts of components for a particular vaccine can be determined through standard clinical trials involving observation of an appropriate immune response in a subject. For example, in another embodiment, the administration of human vaccination Amounts are determined by extrapolating from animal studies to human data. Dosage is determined empirically.

[0087] An "effective amount" of a composition of the present invention is an amount of a substance that is effective to inhibit the growth of a microorganism, such as S. nucleus, during a subsequent challenge. The dose required to induce antibodies that significantly reduce the likelihood or severity of Monier's infection Point.

[0088] The methods of the present 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 can be used to prevent and / or alleviate the primary clinical syndrome caused by the

[0089] Administration of the compositions of the present invention may 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 this.)

[0090] The amount of conjugate in each vaccine dose was determined 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 contain 0.1 to 10 μg of the active ingredient, more specifically 1 to 5 μg of the active ingredient. is 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.

[0091] The optimal amounts of components for a particular vaccine can be determined through standard clinical trials involving observation of an appropriate immune response in a subject. For example, in another embodiment, the administration of human vaccination Amounts are determined by extrapolating from animal studies to human data. Dosage is determined empirically.

[0092] 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 The dose of aluminum salt is per μg of recombinant protein.

[0093] Generally, each 0.5 mL dose contains 2 μg of each S. nucleoside except for serotype 6B polysaccharide, which is 4 μg. -monier polysaccharides, approximately 32 μg CRM197 carrier protein (e.g., 32 μg ± 5 μg, ±3 μg, ±2 μg or ±1 μg), 0.125 mg 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, 150mM ± 25mM, ± 20mM, ± 15mM, ± 10mM or ± 5mM). and about 20 mM (e.g., 20 mM ± 5 mM, ± 2.5 mM, ± 2 mM, ± 1 mM or ±0.5 mM) L-histidine buffer.

[0094] According to any of the methods of the present invention, in one embodiment, the subject is a human. In this context, human patients may be infants (under 1 year of age), toddlers (approximately 12-24 months) or young children. 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.

[0095] 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, appropriately spaced apart. 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 based on the schedule 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 animals aged 2, 4, 6 and 12-15 months. It is administered as a four-dose series at 100 mg / kg / day.

[0096] The compositions of the present invention may also include one or more proteins derived from S. pneumoniae. Examples of S. pneumoniae proteins suitable for incorporation include those described in International Patent Application Publication No. WO02 / 026666. 083855 and those identified in International Patent Application Publication No. WO 02 / 053761. nothing.

[0097] formulation The compositions of the present invention can be administered parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, intradermally, or intradermally. The compound can be administered to a subject by one or more methods known to those skilled in the art, such as intravenously, intraperitoneally, or intraperitoneally. It can be formulated accordingly.

[0098] In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous injection, intra-arterial injection, or the like in a liquid formulation. It is administered via intravenous, subcutaneous or intramucosal injection into the respiratory tract. Including etc.

[0099] The compositions of the present invention may be packaged in single dose vials, multi-dose vials or pre-filled syringes. It can be formulated as follows.

[0100] In another embodiment, the compositions of the present invention are administered orally and therefore may be in a form suitable for oral administration. Solid oral formulations include tablets, capsules, and capsules. Liquid oral formulations include solutions, suspensions, dispersions, etc. This includes liquids, emulsions, oils, etc.

[0101] 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, 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

[0102] Pharmaceutical compositions may be isotonic, hypotonic or hypertonic. It is often preferred that pharmaceutical compositions be essentially isotonic upon administration. The pharmaceutical composition may preferably be isotonic or hypertonic for storage. If the solution is hypertonic due to its nature, it can be diluted to an isotonic solution before administration.

[0103] The isotonic agent may be an ionic isotonic agent, such as a salt, or a non-ionic isotonic agent, such as a carbohydrate. Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl2, KC Examples of non-ionic isotonic agents include, but are not limited to, sucrose, PEG-40 ... Contains cellulose, trehalose, mannitol, sorbitol and glycerol.

[0104] Also preferably, the at least one pharmaceutically 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 the solution to a pH in the range of 4 to 10, e.g., 5 to 9, e.g., 6 to 8. It is often desirable to include a buffer containing

[0105] Buffers include, for example, Tris, acetate, glutamate, lactate, maleate, Thalate, phosphate, citrate, carbonate, glycinate, L-histidine glycine, succinate and triethanolamine buffers. do.

[0106] The buffer may, for example, comply with 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.

[0107] 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 dextromethorphan, and fixed oils. Examples include electrolyte replacement fluids such as those based on tallow. Such as water and oils, with or without the addition of other pharmaceutically acceptable adjuvants. It is a sterile liquid. Generally, it is made up of water, saline, aqueous dextrose and related sugar solutions, glycol 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, such as peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, Another fish oil, or a lipid derived from milk or eggs.

[0108] The formulation may also contain a surfactant. Preferred surfactants include, but are not limited to: Although there is no polyoxyethylene sorbitan ester surfactant (commonly called Tween), (used in the art), 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, octoxy(oxy-1,2-ethanediyl) with a variable number of groups Cetoxynol-9 (Triton X-100 or t-octylphenoxypolyethylene glycol) (Octylphenoxy)polyethoxyethanol (I) is of particular interest; GEPAL CA-630 / NP-40; phospholipids, e.g., phosphatidylcholine ( lecithin; nonylphenol ethoxylates, such as Tergitol™ NP Series: Lauryl, Cetyl, Stearyl and Oleyl Alcohols (Brij Surfactants Polyoxyethylene fatty ethers derived from ethylene glycol ethers (known as ethylene glycol ethers), e.g., triethylene glycol ethers glycol monolauryl ether (Brij 30); and sorbitan ester ( commonly known as SPAN), e.g., sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred for inclusion in emulsions The surfactant is PS-20 or PS-80.

[0109] Mixtures of surfactants can be used, for example, PS-80 / Span 85 mixtures. Polyoxyethylene sorbitan monooleate (PS-80) Ethylene sorbitan ester and t-octylphenoxypolyethoxyethanol (Tri Combinations with octoxynol such as methyl ... The combination is laureth 9 + polyoxyethylene sorbitan ester and / or octoxy Including Nol.

[0110] A preferred amount of surfactant is polyoxyethylene sorbitan ester (e.g., PS-80). ) 0.01 to 1% w / v, especially about 0.1% w / v; octyl or nonylphenoxypoly Oxyethanol (Triton X-100, or other washes in the Triton series) agents) 0.001-0.1% w / v, especially 0.005-0.02% w / v; polyoxyethylene Ethylene ether (such as laureth 9) 0.1 to 20% w / v, preferably 0.1 to 10% w / v, particularly 0.1-1% w / v or about 0.5% w / v.

[0111] In certain embodiments, the composition essentially contains 250 μg / mL of APA (aluminum phosphate). L-histidine (20 mM), saline (150 mM), and 0.2% w / v PS-20 at pH 5.8. The presence of PS-20 or PS-80 in the formulation may range from 0.1% w / v to 1.0% w / v. Control aggregation during simulation and during shipping using primary packaging. The process Up to 44 S. pneumoniae polysaccharides in L-histidine, sodium chloride, and PS-20 Combining a blend of somatic serotypes and then incubating this blended material with APA and and sodium chloride, with or without an antimicrobial preservative.

[0112] 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 influences the formulation. In particular, the stability of aqueous or DM Pneumococcal polysaccharide-protein conjugates prepared in SO solvent and combined in multivalent compositions The duplicates exhibit significant differences in stability depending on the particular surfactant system used in the formulation.

[0113] In the formulations described herein, the poloxamer generally ranges from 1,100 Da to 17,400 Da. Da, 7,500Da to 15,000Da or 7,500Da to 10,000Da The poloxamer has a molecular weight of 188 or 407. The final concentration of poloxamer in the formulation of the present invention can be selected from the following. % w / v or 0.025-1% w / v. Poloxamer-containing surfactant systems In certain embodiments, the polyol is propylene glycol. In certain embodiments, the polyol is a polyol at a final concentration of 1-20% w / v. Ethylene glycol 400, at a final concentration of 1–20% w / v.

[0114] 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 approximately 2,700 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.

[0115] The formulation also contains a pH buffered saline solution. The buffer 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 ranges of 4-10, 5.2-7.5, or 5.8-7.0. In certain embodiments, phosphate, succinate, L-histidine, MES, MOP The buffer is selected from the group consisting of HCl, ... The buffer may be, for example, a USP compatible solution for parenteral use, particularly if the pharmaceutical formulation is for parenteral use. The concentration of the buffer may be selected from the group consisting of 1 mM to 50 mM and 5 mM to 50 mM. In certain embodiments, the buffer contains L-His at a final concentration of 5 mM to 50 mM. In certain embodiments, the final concentration of L- Histidine is at a final concentration of 20 mM ± 2 mM.

[0116] Physiological 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, are 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, optionally 2 mM to 170 mM. It may be present at a concentration of 0 mM to 170 mM.

[0117] In a preferred embodiment, the formulation comprises an L-histidine buffer with sodium chloride. .

[0118] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. It can be administered using intravenous infusion, transdermal patch, liposomes, or other modes of administration. In another embodiment, the polymeric material is used, for example, in microspheres or implants. can be.

[0119] The compositions of the present invention may also include one or more proteins derived from S. pneumoniae. Examples of S. pneumoniae proteins suitable for incorporation include those described in International Patent Application Publication No. WO02 / 026666. 083855 and those identified in International Patent Application Publication No. WO 02 / 053761. nothing.

[0120] Analysis method Conjugate molecular weight and Concentration analysis Conjugate samples were separated by high performance size exclusion chromatography (HPSEC). Ultraviolet (UV), Multi-Angle Light Scattering (MALS) and Refractive Index (RI) Detection is achieved using sequential detectors: UV280 to protein using extinction coefficients. Concentration is calculated by the change in refractive index of the solution with a change in solute concentration reported in mL / g. Using certain dn / dc factors, 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 the University of California, 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 (Molecule s, 2015, 20:10313-10341). The term "molecular weight" used throughout is the weight average molecular weight.

[0121] Conjugated saccharide 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 of the conjugate samples, Waters A ccQ-Tag amino acid analysis (AAA) is used. The sample was hydrolyzed using gas phase acid hydrolysis to separate the carrier proteins into their constituents. Decomposes into amino acids. 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate Free amino acids are derivatized using methyl amine (AQC). Derivatized samples were analyzed using UPLC with UV detection. Use appropriate amino acids to obtain average protein concentrations. (i.e., lysine loss) is the ratio of the average measured lysine amount in the conjugate to the predicted amount in the starting protein. It is determined by the difference between the measured lysine amount.

[0122] Free polysaccharide test Free polysaccharides in the conjugate sample (i.e., conjugated with CRM197) The undigested polysaccharides were combined with free proteins and deoxycholate (DOC) and hydrochloric acid. The conjugate is measured by first precipitating the conjugate. The precipitate is then filtered and The filtrate is analyzed for free polysaccharide concentration by HPSEC / UV / MALS / RI. Free polysaccharides were calculated as a percentage of total polysaccharides measured by HPSEC / UV / MALS / RI. It is calculated as follows.

[0123] Free Protein Test Capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode was used to Free polysaccharide, polysaccharide-CRM197 conjugate, and free CR in the conjugate sample Briefly, samples were diluted in 25 mM borate, 100 mM SDS, pH 9. Mix with MEKC running buffer containing 0.3 and preconditioned bare melt sheet. 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 a percentage of total protein content determined by the LS / RI procedure.

[0124] 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 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.

[0125] The following examples illustrate but do not limit the invention.

[0126] [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, e.g., J. Med. Soc. 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP0497524B1. The process generally follows the method described in European Patent No. EP0497524B1, are generally applicable to all pneumococcal serotypes except as modified in

[0127] Pneumococcal subtype 23A and 23F isolates were cultured using Merck Culture Collection (MCL). Serotype 23B strains were obtained from the Centers for Disease Control and Prevention (Atlantic). If necessary, the Quelling reaction was carried out using specific antisera. Subtypes can be distinguished based on the following: See the FDA website for more information on soy peptone, yeast extract, and hemin-free glucose (hemin-free glucose). Agar plates made from animal component-free medium containing serotype 23F (excluding serotype 23F containing serotype 23F) The resulting isolates were further clonally isolated by successive plating on 100 cultures in two steps. Soybean peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate Liquid culture using an animal component-free medium containing sodium, glucose, and glycerol The clonal isolates of each serotype were further expanded to prepare a pre-master cell bank. .

[0128] The production of each serotype of pneumococcal polysaccharide involves cell growth and batch production fermentation followed by downstream purification. For serotypes other than 23F, the inactivation was performed with soybean peptone or Soybean peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride Pre-sterilized containing sodium bicarbonate, potassium phosphate, and glucose Shake flasks or culture bottles containing animal component-free growth medium were used to culture the strains from each serotype. A vial of thawed cell bank was grown in a sealed shake flask or bottle. Cell growth cultures were grown to minimize gas exchange by temperature and agitation control. In Type 23F, a fermenter containing the same medium was used to expand a vial of the thawed cell bank. During cell growth of serotype 23F, temperature, pH, pressure and agitation were controlled. Since no jigs were used, the airflow overlay was also Controlled.

[0129] After achieving a certain culture density, as measured by optical density at 600 nm, cells were cultured. A portion of the cell growth culture is added to soy peptone or soy peptone ultrafiltrate, yeast extract or A pre-mixture containing yeast extract ultrafiltrate, sodium chloride, potassium phosphate, and glucose The culture was transferred to a production fermenter containing sterilized animal component-free growth medium. Temperature, pH, pressure and Agitation was controlled. No sparging was used, so an airflow overlay (Airflo w overlay) was also controlled.

[0130] When glucose is nearly depleted, the buffer is depleted 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 under continuous temperature and agitation conditions to release the capsular polysaccharide from the cell wall. This occurs over a specific time period in a fermenter that is controlled by a centrifuge. After primary inactivation, the batch is transferred to a separate vessel. There, the mixture is further 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 time of inactivation. The inactivated broth was then purified.

[0131] Purification of Ps The purification of pneumococcal polysaccharides involves several steps: centrifugation, depth filtration, concentration / diafiltration trays. All procedures were performed at room temperature unless otherwise specified. Ta.

[0132] Cationic polymer (BPA-1000, Petrolite "Tretolite") and "Spectrum 8160" and poly(ethyleneimine), "Millip ore pDADMAC) to inactivate S. pneumoniae from fermenter cultures. The cationic polymer flocculated the purified broth, removing impurities such as proteins, nucleic acids, and cell debris. After the flocculation step and maturation period, the mixture was separated by centrifugation and multiple depth filtration steps. The aggregated solids were removed. The purified broth was concentrated to a MW of 100 kDa to 500 kDa. Diafiltration was performed using a CO (molecular weight cut-off) filter. Diafiltration using MgCl2 buffer and sodium phosphate buffer Diafiltration removed residual nucleic acids and proteins.

[0133] Additionally, sodium acetate and denatured alcohol and / or isopropanol Impurities were removed by reprecipitation of the polysaccharide in phenol. The diafiltered retentate was diluted with sodium acetate in sodium phosphate saline buffer. The thorium and phenol (liquid phenol or solid phenol) were added. The alcohol fractionation of 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. The polysaccharides are recovered from the solution by adding propanol or denatured alcohol to the batch. The precipitated polysaccharide pellet was collected by centrifugation, crushed, and dried to a powder. The cells were stored frozen at -70°C.

[0134] [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, JAI Press Inc. The purified polysaccharide was analyzed using standard 1D and 2D NMR techniques. Finally, we investigated the glycosides. 31 P NMR was used to characterize the polysaccharide for the presence of phosphate. The body was examined.

[0135] 1 H- 1 H COSY, double quantum filtered kernel 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 (HES). eronuclear single quantum coherence spec HSQC (High-Speed ​​Quantitative Chemistry), 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.

[0136] 1D phosphorus NMR spectroscopy reveals that S. pneumoniae serotype 23A and 23B polysaccharides are structurally It was shown that it contains phosphorus. 1 H- 31 Assignment of phosphorus binding sites was determined by P HMBC. Ta.

[0137] Based on the NMR data in Figures 2 to 5, the structure of the S. pneumoniae serotype 23A 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.

[0138] Based on the NMR data in Figures 2 to 5, the structure of the S. pneumoniae serotype 23B 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 1B.

[0139] The sugar residues of S. pneumoniae serotype 23A and 23B polysaccharides are rhamnose (Rha). , galactose (Gal), glucose (Glc) and glycerol.

[0140] The italics (p and f) indicate pyranoses (closed rings of six atoms) and furanoses. It refers to a 5-atom closed ring.

[0141] 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). β means that the α-proton is in the equatorial position in the 3D structure. This means that the ton is in the axial position.

[0142] 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 Rhamno. This means that the first carbon of glucose is bonded to the third carbon of glucose (p means that these (This also means that it is a pyranose ring).

[0143] Identification of the activation site Aldehydes (usually hydrated) are reacted with thiosemicarbazide (T) in 5 mM citrate buffer. The activation site was identified by reacting TSC with aldehydes (and water). The resulting imine reacts with the aldehyde to form an imine (secondary aldimine). The imine has a unique chemical shift that is downfield of the polysaccharide signal. Proton was used to investigate the oxidation sites of polysaccharides.

[0144] The oxidized S. pneumoniae serotype 23B polysaccharide was diluted with sodium citrate buffer and then with thiosemicarbazide at ambient temperature, followed by continuous mixing and freeze-drying. For NMR analysis, the freeze-dried sample was dissolved in 0.9 mL of deuterium oxide.

[0145] Using a cryogenically cooled probe, NM at 600 MHz at a probe temperature of 25 °C R experiment was performed. 16 transient 90-degree pulses with a 10-second delay between pulses (acquisition time). 1D proton spectra were acquired using a 4-transistor setup (including a 3-second delay). in the second dimension by 256 and 512 increments, respectively. The gradient COSY data were acquired: 16 transients in the first dimension and 2 in the second dimension. NOESY data were acquired in 56 increments.

[0146] After TSC derivatization, all activated aldehydes were converted to imines. The chemical shift of the protons of hydride shifted to 7-8 ppm (Figure 6). Two groups of peaks were observed between 7.28 pm and 7.5 ppm (7.28 ppm and 7.36–7.4 ppm). 2D TOCSY indicated that 7.36-7.40ppm The correlation between the m peak and the rhamnose CH3 peak (approximately 1.32 ppm; Figure 7) was observed. The peak (7.36-7.40 ppm) corresponds to the proton chain of TSC-derivatized 23B rhamnose. The structure of the non-activated serotype 23B polysaccharide suggested that the rhamnose ring The only possible proton in the upper 7.36-7.40 ppm peak is H3.

[0147] The COSY data show that the peak at 5.46 ppm correlates with the peak at 7.28 ppm. The NOESY data showed that the peak at 5.46 ppm was a peak at 7.37 ppm (H3). These data indicated that the peak at 5.46 ppm was in close proximity to the TS peak. The peak at 7.28 ppm belongs to C-derivatized rhamnose H1, and the peak at 7.28 ppm belongs to TSC-derivatized rhamnose. This suggests that this belongs to H2 (Figure 8).

[0148] According to the above data, the main activation site of serotype 23B polysaccharide is shown in Figure 1B. Rhamnose is located at C2 / C3 position.

[0149] [Example 3] S. pneumoniae to CRM197 using reductive amination in dimethyl sulfoxide Conjugation of E. coli serotype 23A polysaccharide The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and diluted by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and then lyophilized. The redissolved polysaccharide and CR were then redissolved in dimethyl sulfoxide (DMSO). The M197 solutions were combined and conjugated as follows: Final 0.2 microns The resulting conjugate was purified by ultrafiltration before filtration. Several process parameters within each step, such as temperature and time, are controlled to achieve the desired attributes. A conjugate having the formula:

[0150] 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 reduced by acid hydrolysis or homogenization. Add 100 ml of cold potassium phosphate buffer (pH 7) and incubate at 90°C for 1.5 hours. Acid hydrolysis was carried out by adding HCl to the solution to neutralize it to 400 mM. The pressure and the number of passes through the homogenizer were controlled to 800-1000 bar / 5 passes. .

[0151] The size-reduced polysaccharides were concentrated and purified using a 5 NMWCO tangential flow ultrafiltration membrane. The solution was diafiltered against

[0152] The polysaccharide solution was then adjusted to 22°C and pH 5 using sodium acetate buffer. The size reduction of polysaccharides due to activation was minimized. The activation of polysaccharides was initiated by adding the ammonium solution. To achieve this, the added sodium metaperiodate is added in an amount equivalent to 1 mole of polysaccharide repeating unit. The amount of sodium metaperiodate per unit area was 0.20-0.24 mol (polysaccharide repeating unit). The oxidation reaction proceeded for 2 hours at 22°C.

[0153] Diafiltrate the activated product against 10 mM potassium phosphate, pH 6.4. followed by dilution against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0154] 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 purified by filtration. Diafiltration was performed using a filtration membrane against 2 mM phosphate, pH 7.0 buffer. The solution was filtered at 0.2 microns.

[0155] Activated polysaccharide lyophilized at 6 mg Ps / mL with a sucrose concentration of 5% w / v CRM197 was formulated for 6 mg Pr / mL and formulated for lyophilization.

[0156] 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 concentration of 25-50 mM. The polysaccharide concentration is 1.8-3.0 g Ps / L (grams of polysaccharide per liter) and The mass ratio of polysaccharide to CRM197 was 1.5. The ratio of polysaccharide to CRM197 in the conjugate was controlled. Add 1 mole of glycerol (1 mole per mole of polysaccharide repeating unit) and let the conjugate stand at 22°C for 2-4 hours. The project progressed.

[0157] 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 to 3 hours. Dilute the batch with 150 mM sodium chloride containing 25% (w / v) polysorbate 20. Then, potassium phosphate buffer was added to neutralize the pH. Concentrate the batch and filter the 150 mM salt solution using a 30 kD NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed at approximately 4°C against 25 mM sodium chloride and 25 mM potassium phosphate pH 7. I did.

[0158] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to 0.05 ml. in 150 mM sodium chloride pH 7.0 containing 0.015% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine at 4°C.

[0159] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. 10 mM histidine in additional 150 mM sodium chloride pH 7.0 containing 20 Diluted, aliquoted and frozen at ≦-60°C.

[0160] Table 1 shows the properties of the serotype 23A conjugate prepared in DMSO. [Table 1]

[0161] [Example 4] S. pneumoniae to CRM197 using reductive amination in dimethyl sulfoxide Conjugation of E. coli serotype 23B polysaccharide The polysaccharides are dissolved, sized to the target molecular weight, chemically activated, and diluted by ultrafiltration. The activated polysaccharide and purified CRM197 were separately lyophilized and then lyophilized. The redissolved polysaccharide and CR were then redissolved in dimethyl sulfoxide (DMSO). The M197 solutions were combined and conjugated as follows: Final 0.2 microns The resulting conjugate was purified by ultrafiltration before filtration. Several process parameters within each step, such as temperature and time, are controlled to obtain the desired attributes. The conjugates were obtained with good activity.

[0162] Polysaccharide size reduction and oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 microns. The polysaccharide was homogenized to reduce the molecular weight of Ps. The number of passes was controlled at 400 bar / 5 passes.

[0163] Size-reduced polysaccharides are concentrated using a 10 kDa NMWCO tangential flow ultrafiltration membrane. and diafiltered against water.

[0164] The polysaccharide solution was then adjusted to 22°C and pH 5 using sodium acetate buffer. The size reduction of polysaccharides due to activation was minimized. The activation of polysaccharides was initiated by adding the ammonium solution. To achieve this, 0.10 moles of sodium metaperiodate per mole of polysaccharide repeat unit were used. Sodium metaperiodate was added at 0.13 moles (per mole of polysaccharide repeating unit). (number of moles of aldehyde per mole).

[0165] Diafiltrate the activated product against 10 mM potassium phosphate, pH 6.4. followed by dialysis against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2-8°C.

[0166] 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 purified by filtration. Diafiltration was performed using a filtration membrane against 2 mM phosphate, pH 7.0 buffer. The solution was filtered at 0.2 microns.

[0167] Activated polysaccharide lyophilized at 6 mg Ps / mL with a sucrose concentration of 5% w / v CRM197 was formulated for 6 mg Pr / mL and formulated for lyophilization.

[0168] 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 0-50 mM. The polysaccharide concentration was 5.0 g Ps / L and the mass ratio of polysaccharide to CRM197 was 1. The mass ratio of polysaccharide to CRM197 in the resulting conjugate was selected to achieve 0.5. The ratio of sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was controlled. mol) was added and conjugation was allowed to proceed at 22°C for 2-4 hours.

[0169] 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. The batch was diluted in 150 mM sodium chloride containing % (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. The batch was concentrated to a concentration of 30 kDa N Using a MWCO tangential flow ultrafiltration membrane, 150 mM sodium chloride, 25 mM potassium phosphate The solution was diafiltered against HCl pH 7 at approximately 4°C.

[0170] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane to 0.05 ml. in 150 mM sodium chloride pH 7.0 containing 0.015% (w / v) polysorbate 20 The solution was diafiltered against 10 mM histidine at 4°C.

[0171] The retentate batch was filtered at 0.2 microns and then diluted with 0.015% (w / v) Polysorbate. 10 mM histidine in additional 150 mM sodium chloride pH 7.0 containing 20 Diluted, aliquoted and frozen at ≦-60°C.

[0172] Table 2 shows S. pneumoniae serotype 23B polysaccharide conjugates prepared in DMSO. Indicates the attribute of. [Table 2]

[0173] [Example 5] Formulation of monovalent conjugates Pneumococci from serotypes 23A and 23B, as described in Examples 3 and 4 Polysaccharide-CRM197 conjugates were prepared as described in U.S. Pat. No. 8,192,746. As described, pneumococcal polysaccharide-CRM197 conjugate from serotype 23F Based on the batch volume and concentration of each bulk polysaccharide concentration, individual serotypes were prepared. The required amount of bulk conjugate needed to achieve the target concentration of S. pneumoniae was calculated. Bulk conjugates of E serotypes (23A, 23B, and 23F) were combined with excipients. , sterile filtered, and added to APA under mixed conditions. 20 mM histidine, 150 mM NaCl l, 0.2% (w / v) PS-20, and 0.250 mg / mL (w / The final concentration of each monovalent conjugate vaccine containing 4 μg / mL (w / v P nPs).

[0174] [Example 6] 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 injected intramuscularly with 0.25 ml of ethanol. The animals were immunized intramuscularly (IM). 62.5 μg of aluminum phosphate adjuvant was used per immunization. 1 μg of PnPs (each conjugated to CRM197) was administered together with APA. monovalent pneumococcal conjugates at doses of S. pneumoniae serotype 23A or 23B polysaccharide The vaccine was administered before the start of the study (pre-immunization) and on day 14 (post-dose 1, PD1) and Serum was collected on days 28 and 29 (post-dose 2, PD2). NZWR were observed at least daily by trained animal care staff. The cutin preparation was deemed safe and well tolerated. All animal studies were conducted by the National Institutes of Health. Guide for Care and Use of Laboratory Ani The NZWR experimental protocol was carried out in strict accordance with the recommendations of Merck & Co. o., Inc. (Kenilworth, NJ) and Covance (Denver, PA) A) Institutional Animal Care and Use Center Approved by mittees.

[0175] ELISA of NZWR serum using each PnPs coating concentration of 1–2 mg / ml The immunogenicity of the IgG was evaluated by opsonization assay based on the previously described protocol. Functional antibodies were determined by immunophagocytosis assay (OPA). guilar et al.,2017,Vaccine 35:865-72 and B Urton et al.,2006,Clin Vaccine Immunol 1 3(9):1004-9.

[0176] Monovalent pneumococcal polysaccharide conjugates from S. pneumoniae serotypes 23A and 23B The vaccine was immunogenic in rabbits (Figure 9) and produced functional antibodies that killed each bacterial strain. It was found to produce (Figure 10).

[0177] [Example 7] Monovalent conjugate New Zealand white rabbit immunogenicity study (23A, 23B, 23F) Cross protection) 23A-CRM197 / APA, 23B-CRM197 / APA or 23F-CRM Rabbits were immunized with 197 / APA to examine cross-reactivity between each S. pneumoniae serotype. Evaluated.

[0178] Overall, immunization with the S. pneumoniae serogroup 23 monovalent conjugate vaccine In rabbits, IgG and OPA titers were increased against the homologous polysaccharide and bacterial strain, respectively. was the highest (Figures 11A-11B, Figures 12A-12B, Figures 13A-13B). 3A-CRM197 / APA and 23F-CRM197 / APA are 23B-CRM1 Compared with rabbits immunized with the 97 / APA conjugate, S. pneumoniae 23 Low or no cross-reactivity to B serogroup PnPs and bacterial strains Using Dunnett's multiple comparison test, 23B-CRM1 In 97 / APA-immunized rabbits, 23A-CRM197 / APA and 23F-CRM19 IgG immunogenicity was significantly higher compared with 7 / APA-immunized rabbits (P = 0. Similarly, the 23B-CRM197 / APA immune cells Herons were immunized with 23A-CRM197 / APA and 23F-CRM197 / APA rabbits. had significantly higher functional antibodies compared with the control group (P = 0.0002 and 0.00 2) (Figure 12B). To cover S. pneumoniae serogroup 23, the pneumococcal polysaccharide component The adjugate vaccine protects against S. pneumoniae serotypes 23A, 23B, and 23F. It should contain at least serotype 23A / 23F polysaccharide and serotype 23B polysaccharide to be.

[0179] [Example 8] Formulation of pneumococcal conjugate vaccines for rabbit polyvalent testing. Pneumococcal polysaccharide-CRM197 conjugates were used to evaluate the efficacy of different conjugate ballasts. Cuvrend preparation (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 84 μg / mL of serotype 4 μg / mL of each serotype, 20 mM histidine pH 5.8 and and 150 mM sodium chloride and 0.1% w / v polysorbate-20 (PS-20 ) was formulated with pneumococcal polysaccharide (PnPs) types (S. pneumoniae serotypes 16F, 23 CRM197 protein was individually cloned into the 23A, 23B, 24F, and 31 Conjugates were prepared by conjugating individual bulk polysaccharide concentrations. Based on the volume and concentration of each serotype, the amount of bulk conjugate required to achieve the target concentration of each serotype was determined. The required amount of gates was calculated. Each conjugate was mixed with histidine, sodium chloride, and and polysorbate-20 (PS-20) solutions to form a conjugate blend. The formulation vessel containing the conjugate blend was mixed using a magnetic stir bar and sterile filtered. The formulation was then transferred into a plastic syringe, glass syringe or The solution was then filled into vials and stored at 2 to 8°C.

[0180] [Example 9] Immunogenicity of a polyvalent pneumococcal conjugate vaccine in New Zealand White rabbits On days 0 and 14 (alternating sides), the multivalent pneumococcal conjugate described in Example 8 was administered. Adult New Zealand White rabbits (NZWR, n=5 / 10) were vaccinated with 0.5 ml of the vaccine. Group 1) was immunized intramuscularly (IM). Each conjugate PnPs was administered in a volume of 2 μg per immunization. The multivalent pneumococcal conjugate vaccine was administered at 14 days after the start of the study (pre-immunization). Serum was collected on days 1 (post-dose 1, PD1) and 28 (post-dose 2, PD2). or signs of distress, and trained animal care staff administer NZWR to the animals at least daily. The vaccine formulation against NZWR was considered safe and well tolerated. All studies were conducted in accordance with the National Institutes of Health's Guide for Care and Use of All experiments were carried out in strict accordance with the recommendations for Laboratory Animals. The experimental protocol was developed by Merck & Co., Inc. and Covance (Denver, PA). A) Institutional Animal Care and Use Center Approved by mittees.

[0181] Multiplexed electrochemiluminescence Evaluating IgG immunogenicity of NZWR sera using an electron capture luminescence (ECL) assay This assay uses SULFO-TAG™ labels that emit light upon electrochemical stimulation. MesoScale Discovery (MesoScale Diag) Developed by NASA, a division of NASA, Inc., Gaithersburg, MD Using this technique, Marchese et al. d validation of a multiplex, electrochemi luminescence-based detection assay for t he quantitation of immunoglobulin G sero type-specific antipneumococcal antibodie s in human serum.Clin Vaccine Immunol.16 (3):387-96(2009)) based on the human assay. Developed for use with guinea pig serum as a secondary antibody for testing NZWR serum samples SULFO-TAG™-labeled anti-rabbit IgG was used. (Trademark) 3 software (UAB Research Foundation, Caro- Aguilar et al., 2017, see above; Burton et al., 20 06, see above) from the University of Alabama at Birmingham, using Bacterial R respiratory Pathogen Reference Laboratory Based on the aforementioned protocol available online at Functional antibodies were determined by sonophagocytic assays (MOPA). Ta.

[0182] S. pneumoniae serotypes 16F, 23A, in multivalent pneumococcal conjugate vaccines Polysaccharide-protein conjugates prepared from 23B, 24F, and 31 showed immunogenic in rabbits at both post-dose 1 (PD1) and post-dose 2 (PD2) These were also found to produce functional antibodies that killed vaccine-type bacterial strains. Immunized with a 2 μg dose of multivalent pneumococcal conjugate vaccine (Figure 15). In rabbits treated with immunization, the MOP of PD1 was significantly higher for the four serotypes compared to pre-immune rabbit serum. A titer was significantly higher (Figure 15). In rabbits immunized with HIV, the five serotypes were significantly higher than in pre-immune rabbit sera. The MOPA titer of PD2 was also significantly higher than that of PD2 (Figure 15). Log-transformed data were analyzed by one-way analysis of variance to determine significance.

Claims

1. An immunogenic composition comprising a polysaccharide-carrier protein conjugate and a pharmaceutically acceptable carrier, Here, the polysaccharide in the polysaccharide-carrier protein conjugate is The polysaccharide comprises serotype 23B of Streptococcus pneumoniae, having a repeating unit of the following structure: 【Chemistry 8】 wherein the carrier protein is CRM197; wherein the polysaccharide-protein conjugate has a molecular weight of 1,000 kDa to 10,000 kDa; and further wherein the polysaccharide-protein conjugate has a polysaccharide-to-protein ratio of 0.5 to 2.0; and wherein the repeating unit of 23B polysaccharide is conjugated at the second or third carbon position of β-Glcp or β-Rhap.

2. further comprising one or more additional polysaccharide-carrier protein conjugates, wherein each conjugate comprises a polysaccharide of a particular Streptococcus pneumoniae serotype conjugated to CRM197; and wherein the serotype of Streptococcus pneumoniae is selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38.

3. 3. The immunogenic composition of claim 2, formulated to contain 0.4 to 4 μg / mL of each polysaccharide, except for serotype 6B polysaccharide, which, if present, contains 0.8 to 8 μg / mL of polysaccharide, and CRM197 carrier protein in an amount 0.5 to 3 times the total amount of polysaccharide.

4. 4. The immunogenic composition of claim 3, further comprising 150 mM sodium chloride, 20 mM L-histidine buffer, and 0.05 to 2% w / v surfactant.

5. The immunogenic composition of claim 4, further comprising an adjuvant.

6. The immunogenic composition of claim 5, wherein the adjuvant is an aluminum-based adjuvant.

7. 7. The immunogenic composition of claim 6, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.

8. The immunogenic composition of claim 7, wherein the adjuvant is aluminum phosphate.

9. 10. The immunogenic composition of claim 2 for preventing a Streptococcus pneumoniae infection, disease, or condition associated with serotype 23F S. pneumoniae in a subject.

10. 2. The immunogenic composition of claim 1, wherein at least 90% of the repeating units of 23B polysaccharide are conjugated at the second or third carbon position of β-Rhap.

11. The immunogenic composition of claim 1, wherein the degree of conjugation of the conjugate is 3 to 13.

12. The immunogenic composition of claim 1, wherein the polysaccharide has a molecular weight of 50 kDa to 1000 kDa.

13. The immunogenic composition of claim 1, wherein the polysaccharide has a molecular weight of 100 kDa to 300 kDa.

14. 2. The immunogenic composition of claim 1, wherein the polysaccharide-carrier protein conjugate has a polysaccharide to carrier protein mass ratio of 0.5 to 1.

5.

15. 2. The immunogenic composition of claim 1, wherein the polysaccharide-carrier protein conjugate has a polysaccharide to carrier protein mass ratio of 0.8 to 1.

2.

16. 2. The immunogenic composition of claim 1, wherein the polysaccharide-carrier protein conjugate comprises less than 25% non-covalently bound polysaccharide compared to the total amount of polysaccharide.

17. 2. The immunogenic composition of claim 1, wherein the polysaccharide-carrier protein conjugate comprises less than 15% non-covalently bound polysaccharide compared to the total amount of polysaccharide.

18. 2. The immunogenic composition of claim 1, wherein at least 90% of the repeating units of 23B polysaccharide are conjugated at the second or third carbon position of β-Rhap; wherein the degree of conjugation of the conjugate is 3 to 13; wherein the polysaccharide has a molecular weight of 100 kDa to 300 kDa; wherein the polysaccharide-protein conjugate has a polysaccharide-to-protein weight ratio of 0.8 to 1.2; and wherein the polysaccharide-carrier protein conjugate comprises less than 15% non-covalently bound polysaccharide compared to the total amount of polysaccharide.

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