Pneumococcal conjugate vaccine preparation

Abstract

To provide a pneumococcus conjugate vaccine formulation including a surfactant system in which polysorbate 20 or a combination of poloxamer and polyol is incorporated.SOLUTION: A pharmaceutical preparation contains: (i) one or a plurality of polysaccharide-protein conjugate body; (ii) pH buffer saline having pH in the range of 5.0 to 7.5; (iii) aluminum salt; and (iv) polysorbate 20 (PS-20), where the polysaccharide is Streptococcus pneumoniae polysaccharide, and 10% or more of the conjugate body (on the total protein base) is prepared in aprotic solvent.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of 17 / 412,550 filed August 26, 2021, which is a continuation of 16 / 487,610 filed August 21, 2019, which is a 371 national stage application of International Application No. PCT / US2018 / 018659, which claims the benefit of U.S. Provisional Application No. 62 / 463,220 filed February 24, 2017, which is incorporated herein by reference in its entirety. The present invention provides pneumococcal conjugate vaccine formulations that include a surfactant system incorporating a combination of polysorbate 20 or a poloxamer and a polyol. [Background technology]

[0002] Streptococcus pneumoniae, an example of an encapsulated bacterium, Occoccus pneumoniae is a significant cause of serious illness worldwide. In 1997, the Centers for Disease Control (CDC) estimated that there were 3,000 cases of pneumococcal disease each year in the United States. meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal encephalopathy otitis media, and an estimated 500,000 cases of pneumococcal pneumonia. for Disease Control and Prevention,MMWR Morb Mortal Wkly Rep 1997,46(RR-8):l-13 Furthermore, these disease complications have been shown in several studies to be associated with pneumococcal meningitis. The mortality rate can be severe, with up to 8% and neurological sequelae reported in 25% of cases. rditi et al., 1998, Pediatrics 102:1087-97 Please refer to.

[0003] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, is used in adults, especially the elderly and has proven invaluable in preventing pneumococcal disease in people at high risk However, infants and young children have a high risk of developing unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell independent immunogens and are associated with poor response to steroids. Induces weak or no response in infants. Chemical conjugation of chlorinated immunogens induces T cell-dependent immune responses in infants Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM 197 the presence of T cell stimulatory epitopes in their amino acid sequences as a carrier protein for bacterial polysaccharide immunogens.

[0004] The seven most frequently isolated blood strains causing invasive pneumococcal disease in infants and toddlers at the time Pneumococcal colonies containing serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) Prevnar®, a vaccinated vaccine, was first marketed in the United States in February 2000. Following the universal use of Prevnar® in the United States, The serotypes present in vnar® have contributed to the reduction of invasive pneumococcal disease in children. The Centers for Disease Control and Prevention d Prevention,MMWR Morb Mortal Wkly Rep 2 005,54(36):893-7. However, in some areas of the world The limitations of serotype coverage with Prevnar® in the United States and There is some evidence of some newly emerged serotypes (e.g., 19A, etc.). O'Brien et al., 2004, Am J Epidemiol 159:634-44, W hitney et al.,2003,N Engl J Med 348:1737 -46, Kyaw et al.,2006,N Engl J Med 354:14 55-63, Hicks et al., 2007, J Infect Dis 196 :1346-54, Traore et al., 2009, Clin Infect See Dis 48:S181-S189.

[0005] Prevnar 13® is a serotype 1, 3, 4, 5, 6A, 6B, 7F, and 9V 13-valent pneumococcal polysaccharides containing 14, 18C, 19A, 19F, and 23F -protein conjugate vaccines. See, for example, U.S. Patent Application Publication No. 2006 / 004494. No. 228380A1, Prymula et al., 2006, Lancet 367 :740-48, and Kieninger et al.,Safety and I mmunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vacci ne Given as a 4-Dose Series in Healthy I nfants and Toddlers,presented at the 48 t h Annual ICAAC / ISDA 46 th Annual Meeting, See Washington DC, October 25-28, 2008. Dagan et al.,1998,Infect Immun.66:2093-2 See also 098 and Fattom, 1999, Vaccine 17:126 stomach.

[0006] Chinese Patent Application Publication No. 101590224A covers serotypes 1, 2, 4, 5, 6A, 6B, 14-valent pneumococcal pneumoniae containing 7F, 9N, 9V, 14, 18C, 19A, 19F, and 23F A polysaccharide-protein conjugate vaccine is described.

[0007] U.S. Patent No. 8,192,746 is a separate CRM 197 Conjugated to polypeptide Gated serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 15-valent pneumococcal polysaccharide-antibody with 19F, 22F, 23F, and 33F Protein conjugate vaccines are described.

[0008] Several carrier protein systems have also been described. See, e.g., U.S. Patent Application Publication No. 201002 No. 09450, No. 20100074922, No. 20090017059, No. 2009 See Nos. 0010959 and 20090017072.

[0009] Streptococcus pneumoniae polysaccharide-protein conjugates Also contains polysorbate 80 (PS-80) and poloxamer 188 (P188) Formulations containing surfactants are disclosed in U.S. Pat. Nos. 8,562,999 and 8,562,999, respectively. and U.S. Patent Application Publication No. 20130273098. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2006 / 0228380A1 [Patent Document 2] Chinese Patent Application Publication No. 101590224A [Patent Document 3] U.S. Patent No. 8,192,746 [Patent Document 4] U.S. Patent Application Publication No. 20100209450 [Patent Document 5] U.S. Patent Application Publication No. 20100074922 [Patent Document 6] U.S. Patent Application Publication No. 20090017059 [Patent Document 7] U.S. Patent Application Publication No. 20090010959 [Patent Document 8] U.S. Patent Application Publication No. 20090017072 [Patent Document 9] U.S. Patent No. 8,562,999 [Patent Document 10] U.S. Patent Application Publication No. 20130273098 [Non-patent literature]

[0011] [Non-Patent Document 1] Centers for Disease Control and Prevention,MMWR Morb Mortal Wkly Rep 1997,46(RR-8):l-13 [Non-patent document 2] Arditi et al., 1998, Pediatrics 102:1087-97 [Non-patent document 3] Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005,54(36):893-7 [Non-patent document 4] O'Brien et al., 2004, Am J Epidemiol 159:634-44 [Non-Patent Document 5] Whitney et al.,2003,N Engl J Med 348:1737-46 [Non-patent document 6] Kyaw et al.,2006,N Engl J Med 354:1455-63 [Non-Patent Document 7] Hicks et al.,2007,J Infect Dis 196:1346-54 [Non-patent document 8] Traore et al., 2009, Clin Infect Dis 48:S181-S189 [Non-Patent Document 9] Prymula et al.,2006,Lancet 367:740-48 [Non-Patent Document 10] Kieninger et al.,Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers,presented at the 48th Annual ICAAC / ISDA 46th Annual Meeting,Washington DC,October 25-28,2008 [Non-Patent Document 11] Dagan et al.,1998,Infect Immun.66:2093-2098 and Fattom,1999,Vaccine 17:126 Summary of the Invention [Means for solving the problem]

[0012] The present invention relates to a method for producing a polysaccharide-protein conjugate comprising: (i) one or more polysaccharide-protein conjugates; (ii) a pH buffered saline solution having a pH in the range of 5.0 to 7.5; and (iii) an aluminum (iv) a) Polysorbate 20 and (b) 1100 Da to 17,400 Da Poloxamers with molecular weights in the range of 0.1 and 1.2, as well as propylene glycol (PG) and poly(propylene glycol). surfactants selected from polyols selected from polyethylene glycol (PEG) 400; and a therapeutic agent system.

[0013] In certain embodiments, one or more of the polysaccharide-protein conjugates is prepared in an aprotic solvent, such as dimethyl sulfoxide (DMSO). In some embodiments, the protein content is 10%, 15%, 20%, 25%, or more (based on total protein). 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more The conjugates are prepared in aprotic solvents such as DMSO. 10-100%, 24%-100%, or 24-80% conjugates (protein basis) The gate entity is prepared in an aprotic solvent, such as DMSO. In this case, the surfactant system may be polysorbate 20 or a poloxamer / polyol combination as described above. It is a combination.

[0014] In one embodiment, the surfactant system has a molecular weight of 1100 Da to 17,400 Da, 7,500 Da molecular weight in the range of 1 to 15,000 Da, or 7,500 Da to 10,000 Da Poloxamers include poloxamer 188 or poloxamer 407. In some embodiments, the final concentration of poloxamer is 0.001% w / v to In certain embodiments, the polyol is Propylene glycol, at a final concentration of 1% w / v to 20% w / v. In an embodiment, the polyol is polyethylene glycol 400, and is present in an amount of 1 w / v% to 20 w / v%. In one embodiment, the surfactant system comprises polysorbate 20. In one embodiment, the final concentration of polysorbate 20 is 0.001 w / v% to 10 w / v% , or 0.025w / v%~2.5w / v%, or 0.025w / v%~0.1w / In some embodiments where the surfactant system includes PS-20, the formulation is in the range of propylene glycol ether. The polyol may further comprise a polyol selected from glycol and polyethylene glycol. Ethylene glycol or propylene glycol should be used at a final concentration of 6 w / v% to 20 w / v%. In some embodiments, the polyethylene glycol may be polyethylene glycol. Recall 400.

[0015] In certain embodiments, the pH buffered saline solution may have a pH in the range of 5.0 to 7.0. The agents are phosphate, succinate, L-histidine, MES, MOPS, HEPES, and acetate. In one embodiment, the buffering agent may be selected from the group consisting of 5 mM to 50 mM citrate. L-histidine at a final concentration of 1 mM, or succinate at a final concentration of 1 mM to 10 mM. In certain embodiments, L-histidine is at a final concentration of 20 mM ± 2 mM. The salt in the brine may be magnesium chloride, potassium chloride, sodium chloride, or a combination thereof. In one embodiment, the pH buffered saline is sodium chloride. It may be present at a concentration of 20 mM to 170 mM.

[0016] In one embodiment, the polysaccharide-protein conjugate is a carrier protein. The antibody comprises one or more pneumococcal polysaccharides conjugated to a substrate. So, the carrier protein is CRM 197 , diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT Fragment C, pertussis toxoid, cholera toxoid, Escherichia coli (E. coli i) LT (heat-labile enterotoxin), Escherichia coli ST (heat-stable enterotoxin) toxin), Pseudomonas aeruginosa In certain embodiments, the polynucleotide is selected from the group consisting of exotoxin A from Polypeptide B (Polypeptide B), Polypeptide C (Polypeptide C), Polypeptide D (Polypeptide E), Polypeptide E ... B (Polypeptide B), Polypeptide C (Polypeptide E), Polypeptide B (Polypeptide B), Polypept One or more of the saccharide-protein conjugates may be a CRM 197 Nikonji In one embodiment, the polysaccharide-protein conjugate One or more are prepared using reductive amination in the non-aqueous solvent DMSO. In embodiments, polyclonal antibodies from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are used. Saccharide-protein conjugates were synthesized using reductive amination in DMSO. and can be prepared from polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F. The carboxylate-protein conjugates were prepared using reductive amination in aqueous solution. In some embodiments, each dose is 8 μg / mL or 16 μg / mL, except for 6B. , 4 μg / mL or 8 μg / mL of each saccharide, and approximately 64 μg / mL or 1 28 μg / mL CRM 197 It is formulated to contain a carrier protein.

[0017] The present invention is a CRM 197 Serotypes 1, 3, 4, and 5 conjugated to polypeptides; 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33 Streptococcus pneumoniae polysaccharides derived from F and 20 mM L-histidine gin, 150 mM NaCl, 0.2% (w / v) PS-20, 250 μg / mL The present invention is also directed to a 15-valent pneumococcal conjugate formulation comprising an APA of , formulations are 4 μg / mL or 8 μg / mL except for 6B, which is 8 μg / mL or 16 μg / mL. / mL of each saccharide and approximately 64 μg / mL or 128 μg / mL of CRM 197 Responsible In one embodiment, the serotypes 6A, 6B, and 6C are formulated into a dosage form containing the serotypes 6A, 6B, and 6C. Polysaccharide-protein complexes from 7F, 18C, 19A, 19F, and 23F The adjugates were prepared under DMSO conditions and were isolated from serotypes 1, 3, 4, 5, 9V, and 14. Polysaccharide-protein conjugates from , 22F, and 33F were prepared in aqueous It is prepared using the conditions: [Brief explanation of the drawings]

[0018] [Figure 1A]Figure 1 shows SDS-PAGE analysis of DTFB process intermediates under non-reducing conditions. A: Samples shown are: molecular weight standard (lane 1 from left), eluate product from triplicate multimodal anion exchange chromatography runs (MM AEX1, MM AEX2, MM AEX3, lanes 2-4), pooled eluate product from multimodal anion exchange chromatography runs (MM AEX pool, lane 5), diafiltered retentate (UF-DR, lane 6), and final bulk intermediate after 0.2 micron filtration (FBI, lane 7). SDS-PAGE: NuPAGE 4-12% Bis-Tris gel, 5 μg / lane, SYPRO Ruby protein gel stain. [Figure 1B] Figure 1 shows SDS-PAGE analysis of DTFB process intermediates under non-reducing conditions. A: SDS-PAGE analysis of DTFB process intermediates run under reducing conditions (NuPAGE 4-12% Bis-Tris gel, lanes 2, 4, 8, and 10: 5 μg / lane, lane 6: 2 μg / lane, SYPRO Ruby protein gel stain). Samples shown are: Mark-12 standard (lanes 1 and 12), purified CRM197 used to produce DTFB (CRM197, lanes 2 and 10), proteolytically cleaved CRM197 loaded onto a multimodal cation exchange chromatography resin following a trypsin digestion step (MM CEX feed, lane 4), the product from multimodal cation exchange chromatography (MM CEX product, lane 6), and the final bulk intermediate after 0.2 micron filtration (DTFB-FBI, lane 8). [Figure 1C]SDS-PAGE analysis of DTFB process intermediates under non-reducing conditions. C: Samples shown are: molecular weight marker (from left, lane 1), initial concentrated retentate following multimodal cation exchange chromatography (ICR, lane 2), diafiltered retentate (UF-DR, lane 3), concentrated retentate after diafiltration (UF-OCR, lane 4), membrane flush for product recovery (UF-W, lane 5), final pooled retentate and flush (UF-FR, lane 6), and final bulk intermediate after 0.2 micron filtration (FBI, lane 6). SDS-PAGE: 14% Tris-glycine gel, 8.3-8.4 μg / lane, GelCode Blue protein gel stain. [Figure 1D] Figure 1 shows SDS-PAGE analysis of DTFB process intermediates under non-reducing conditions.D: SDS-PAGE analysis of DTFB process intermediates run under reducing conditions (NuPAGE 4-12% Bis-Tris gel, SYPRO Ruby protein gel stain). Samples shown are: Mark-12 standard (lanes 1 and 12), proteolytically cleaved CRM197 loaded onto a multimodal cation exchange chromatography resin following a trypsin digestion step (MM CEX feed, lane 2), purified CRM197 used to produce DTFB (CRM197, lane 3), flow-through during column loading (MM CEX flow-through, lane 4), column wash after loading (MM CEX wash, lane 5), product collected after the elution step from multimodal cation exchange chromatography (MM CEX product, lane 7; 10-fold diluted MM CEX product, lane 9), and late-eluted product collected after the elution step from multimodal cation exchange chromatography (late-eluted MM CEX product, lane 11). [Figure 2]Figure 1 shows DTFB protein concentration in 100 mM potassium phosphate (KPi) as a function of pH and sodium chloride (NaCl) concentration. The solutions were kept at room temperature overnight and then centrifuged. The supernatants were assayed by size exclusion chromatography with UV280 absorbance detection. [Figure 3] Figure 1 shows DTFB protein concentration in potassium phosphate (KPi) solutions as a function of polysorbate 20 (PS-20) concentration. The solutions were vortexed at room temperature for 5 minutes and then centrifuged. The supernatants were assayed by size exclusion chromatography with UV280 absorbance detection. [Figure 4] Figure 1 shows ELISA antibody titers for mice immunized with Streptococcus pneumoniae serotype 3 polysaccharide conjugated to either CRM197 or DTFB carrier protein and formulated with aluminum phosphate adjuvant (APA). Mice were immunized with one of two independent serotype 3-CRM197 conjugate lots (lots 1 and 2). [Figure 5] Figure 1 shows survival curves for mice immunized with Streptococcus pneumoniae serotype 3 capsular polysaccharide conjugated to either CRM197 or DTFB carrier protein and formulated with aluminum phosphate adjuvant (APA). Mice were immunized with one of two independent serotype 3-CRM197 conjugate lots (lots 1 and 2). APA or saline-only formulations were also included in the study as controls. Following immunization, mice were then challenged intraperitoneally with serotype 3 bacteria. [Figure 6] Figure 1 shows a laboratory-scale agitation study to evaluate the effect of time and agitation on the particle size distribution of 15-valent pneumococcal polysaccharide (PnPs) conjugate formulations as measured by static light scattering (SLS). All 15 pneumococcal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. The conjugates were formulated in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.25 mg / mL (w / v Al+3) APA for the agitation study. [Figure 7] Figure 1 shows a laboratory-scale agitation study to evaluate the effect of time and agitation on the particle size distribution of a 15-valent pneumococcal polysaccharide conjugate formulation as measured by SLS. All 15 pneumococcal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. The conjugates were formulated in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.25 mg / mL (w / v Al+3) APA with 0.08% w / v or 0.24% w / v poloxamer 188 (P188) for the agitation study. [Figure 8A] Figure 1 shows a laboratory-scale simulated transport and handling study of a 15-valent pneumococcal polysaccharide conjugate formulation in a syringe. All 15 pneumococcal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. The conjugates were formulated in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.25 mg / mL (w / v Al+3) APA without or with 0.2% w / v P188. Particle size distributions measured by SLS before and after 24 hours of horizontal rotation (A) and visual evaluation of the syringes after 24 hours of horizontal rotation (B) are shown. [Figure 8B] Figure 1 shows a laboratory-scale simulated transport and handling study of a 15-valent pneumococcal polysaccharide conjugate formulation in a syringe. All 15 pneumococcal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. The conjugates were formulated in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and 0.25 mg / mL (w / v Al+3) APA without or with 0.2% w / v P188. Particle size distributions measured by SLS before and after 24 hours of horizontal rotation (A) and visual evaluation of the syringes after 24 hours of horizontal rotation (B) are shown. [Figure 9]Figure 1 shows the particle size distribution (expressed as volume-weighted distribution or D[4,3] as measured by SLS) of two 15-valent pneumococcal polysaccharide conjugate formulations with 20 mM L-histidine, pH 5.8, 150 mM NaCl, 0.25 mg / mL (w / v Al+3) APA, and 0.2% w / v P188. Formulation PCV15Aq consisted of pneumococcal polysaccharide-CRM197 conjugates produced by reductive amination in aqueous solution. Formulation PCV15Aq / Non-Aq / ST3-DTFB used a combination of 15 pneumococcal polysaccharide conjugates, some of which were produced by reductive amination in aqueous solution and others by reductive amination in non-aqueous solution. All pneumococcal polysaccharide serotypes in PCV15Aq / Non-Aq / ST3-DTFB were CRM197-conjugated, except for serotype 3 (ST3), which was conjugated to DTFB. The formulations were filled into syringes and agitated horizontally at 4°C for up to 24 hours before SLS evaluation. [Figure 10A] Figure 1 shows D[4,3] values ​​measured by SLS for PCV15Aq / Non-Aq / ST3-DTFB formulations containing P188 (A), PS-80 (B), and PS-20 (A, B) after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 10B] Figure 1 shows D[4,3] values ​​measured by SLS for PCV15Aq / Non-Aq / ST3-DTFB formulations containing P188 (A), PS-80 (B), and PS-20 (A, B) after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 11]This figure shows the concentration of pneumococcal serotype 3-specific IgG from infant rhesus macaques (IRM) immunized with a 15-valent pneumococcal polysaccharide conjugate formulation with aluminum phosphate adjuvant (APA). All formulations contained Streptococcus pneumoniae serotype 3 capsular polysaccharide (ST3) conjugated to CRM197 or DTFB. The PCV15Aq / Non-Aq formulation used a combination of 15 pneumococcal polysaccharide-CRM197 conjugates, some of which were produced by reductive amination in aqueous solutions and others by reductive amination in nonaqueous solutions. The PCV15 formulations contained 0.2 w / v% P188 or 0.1 w / v% PS-20, as indicated. [Figure 12] Serotype 3 OPA (OPK) titers in pups immunized with two 15-valent pneumococcal polysaccharide conjugate vaccines formulated with aluminum phosphate adjuvant (APA) and 0.2% w / v P188. The formulations contained Streptococcus pneumoniae serotype 3 capsular polysaccharide (ST3) conjugated to CRM1 97 (PCV15Ag) or DTFB (PCV15Aq / Non-Aq / ST3-DTFB). [Figure 13] Figure 1 shows particle size distributions measured by SLS for PCV15Aq / Non-Aq / ST3-DTFB formulations after 1 hour of stirring and up to 24 hours of horizontal rotation. The formulations contained 0.2% w / v poloxamer 188 and various concentrations of propylene glycol (PG) or polyethylene glycol 400 (PEG400), as indicated. [Figure 14A]Figure 1 shows a comparative immunogenicity study in pup rhesus macaques of PCV15Aq / Non-AQ / ST3-DTFB formulations containing 0.2 w / v% P188 or 0.1 w / v% PS-20 with 15 w / v% PG, as described in Example 12. Eight animals per group were intramuscularly injected with one of the two formulations at ages T=0 (Dose 1), 1 month (Dose 2), and 2 months (Dose 3). Serum was collected before Dose 1 and 2 weeks after Dose 1, 2, and 3. Serotype-specific IgG concentrations (IgG GMCs) from pre-immunization, post-Dose 1, post-Dose 2, and post-Dose 3 serum samples were measured as described in Example 10. Panel A shows immunogenicity results for serotypes 1, 3, 4, 5, 6A, 6B, 7F, and 9V. Panel B shows immunogenicity results for serotypes 14, 18C, 19A, 19F, 22F, 23F, and 33F. [Figure 14B] Figure 1 shows a comparative immunogenicity study in pup rhesus macaques of PCV15Aq / Non-AQ / ST3-DTFB formulations containing 0.2 w / v% P188 or 0.1 w / v% PS-20 with 15 w / v% PG, as described in Example 12. Eight animals per group were intramuscularly injected with one of the two formulations at ages T=0 (Dose 1), 1 month (Dose 2), and 2 months (Dose 3). Serum was collected before Dose 1 and 2 weeks after Dose 1, 2, and 3. Serotype-specific IgG concentrations (IgG GMCs) from pre-immunization, post-Dose 1, post-Dose 2, and post-Dose 3 serum samples were measured as described in Example 10. Panel A shows immunogenicity results for serotypes 1, 3, 4, 5, 6A, 6B, 7F, and 9V. Panel B shows immunogenicity results for serotypes 14, 18C, 19A, 19F, 22F, 23F, and 33F. [Figure 15A]Figure 14 shows D[4,3] values ​​measured by SLS for PCVAq / Non-Aq formulations described in Example 13 with different percentages of serotypes (Panel A: 24%, Panel B: 50%, Panel C: 62%, Panel D: 79%, and Panel E: 100%) made in DMSO containing 0.05% w / v PS-80, 0.05% w / v PS-20, and 0.2% w / v PS-20 after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 15B] Figure 14 shows D[4,3] values ​​measured by SLS for PCVAq / Non-Aq formulations described in Example 13 with different percentages of serotypes (Panel A: 24%, Panel B: 50%, Panel C: 62%, Panel D: 79%, and Panel E: 100%) made in DMSO containing 0.05% w / v PS-80, 0.05% w / v PS-20, and 0.2% w / v PS-20 after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 15C] Figure 14 shows D[4,3] values ​​measured by SLS for PCVAq / Non-Aq formulations described in Example 13 with different percentages of serotypes (Panel A: 24%, Panel B: 50%, Panel C: 62%, Panel D: 79%, and Panel E: 100%) made in DMSO containing 0.05% w / v PS-80, 0.05% w / v PS-20, and 0.2% w / v PS-20 after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 15D] Figure 14 shows D[4,3] values ​​measured by SLS for PCVAq / Non-Aq formulations described in Example 13 with different percentages of serotypes (Panel A: 24%, Panel B: 50%, Panel C: 62%, Panel D: 79%, and Panel E: 100%) made in DMSO containing 0.05% w / v PS-80, 0.05% w / v PS-20, and 0.2% w / v PS-20 after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 15E]Figure 14 shows D[4,3] values ​​measured by SLS for PCVAq / Non-Aq formulations described in Example 13 with different percentages of serotypes (Panel A: 24%, Panel B: 50%, Panel C: 62%, Panel D: 79%, and Panel E: 100%) made in DMSO containing 0.05% w / v PS-80, 0.05% w / v PS-20, and 0.2% w / v PS-20 after agitation and horizontal rotation in 1.5 mL HyPak syringes for up to 24 hours. [Figure 16] FIG. 1 shows immunogenicity results in New Zealand White rabbits for a 15-valent pneumococcal conjugate formulation in 20 mM histidine pH 5.8, 150 mM NaCl, 250 μg / mL APA, 0.2 w / v% PS-20 with Streptococcus pneumoniae polysaccharides from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugated to CRM197 using reductive amination in DMSO and Streptococcus pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F conjugated to CRM197 using reductive amination in aqueous solution, formulated as a dosage form containing 4 μg / mL of each saccharide except for 6B at 8 μg / mL, and approximately 64 μg / mL of CRM197 carrier protein. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to the use of certain surfactants in multivalent conjugate vaccine formulations, particularly those containing one Alternatively, if multiple conjugates are made in an aprotic solvent such as DMSO, This is due in part to the discovery that the stability of conjugates and their tendency to aggregate can be affected. In addition, some surfactants require the addition of polyols to achieve the necessary stability. requires.

[0020] As used herein, a "protic solvent" refers to a solvent that is free of oxygen (such as in a hydroxyl group). A solvent that has a hydrogen atom attached to it (as in an amine group) or to a nitrogen (as in an amine group). Generally, any solvent containing labile H+ is referred to as a protic solvent.

[0021] As used herein, "aprotic solvent" refers to a polar aprotic solvent. Such solvents lack acidic hydrogen and cannot donate hydrogen. Examples are dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and heptane. Non-aqueous or The term "protic solvent" is used interchangeably with "aprotic solvent." Aprotic solvents may contain, for example, up to 1% , 2%, 5%, 10% or 20% water may be present.

[0022] As used herein, the term "polysaccharides" (Ps) refers to "saccharides" "ides", "oligosaccharides", "polysaccharides", "liposaccharides", "lipooligosaccharides (LOS) ", "lipopolysaccharide (LPS)", "glycosylates", "complex carbohydrates", etc. Any of the compounds commonly used in the immunological and bacterial vaccine arts, including but not limited to It is meant to contain an antigenic saccharide element (or antigenic unit) of the above.

[0023] As used herein, "comprising" when used in conjunction with the immunogenic compositions of the invention The term "consisting of" includes adjuvants and and any other ingredients such as excipients. The term "consisting of" when used in conjunction with adjuvant mixtures refers to those particular Streptococcus pneumoniae has polysaccharide-protein conjugates , other Streptococcus pneumoniae polysaccharide-proteins from different serotypes represents the mixture without the protein conjugate.

[0024] As defined herein, "precipitation," "sediment," "particulate formation," "cloudiness," and The terms "aggregation" and "aggregation" may be used interchangeably and refer to polysaccharide-protein conjugates. It is meant to represent any physical interaction or chemical reaction that results in the agglomeration of gate bodies. The process of aggregation (e.g., protein aggregation) can be influenced by heat, pressure, pH, agitation, shear forces, freeze-thawing, etc. Numerous physical properties including decomposition, dehydration, heavy metals, phenolic compounds, silicone oils, denaturants, etc. It can be caused by chemical stress.

[0025] As defined herein, a "surfactant" of the present invention is an agent that reduces the surface tension of an immunogenic composition formulation. A "surfactant system" is any molecule or compound that reduces the force of a surfactant. However, this may allow for the inclusion of additional excipients such as polyols that enhance the effectiveness of the surfactant.

[0026] The immunogenic compositions of the present invention comprise a medicament comprising a medicament conjugated to one or more carrier proteins. It may be a multivalent composition containing one or more antigens. In such vaccines, the antigen is a saccharide from the encapsulated bacteria. The saccharides consist of long chains of sugar molecules similar to the surfaces of certain bacteria. The bacteria involved were Streptococcus pneumoniae, Neisseria meningitidis (Ne Isseria meningitides), and Haemophilus influenzae b Haemophilus influenzae type b, The antigens may be from the same organism or from different organisms. In another embodiment, the antigen is Streptococcus pneumoniae capsular polysaccharide. It is Do.

[0027] In embodiments where two carrier proteins are used, the first carrier protein is conjugated to the second carrier protein. Each unmodified 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 antibody is not conjugated to a first carrier protein. The capsular polysaccharide is conjugated to two or more carrier proteins (each capsule (Membrane polysaccharide molecules are conjugated to a single carrier protein). In such an embodiment, each capsular polysaccharide of the same serotype is typified by the same carrier protein. are selectively conjugated.

[0028] Corynebacterium diphtheriae Diphtheria toxin, an exotoxin secreted by Diphtheria riae, is formed by disulfide bridges. The classical structure consists of two subunits (fragments) connected by a tether and having three domains. Fragment A (DTFA) is a typical AB toxin. Fragment B (DTFB) contains the central translocated T domain and carboxyl groups. The DTFB contains the C-terminal receptor-binding R domain. Approximately 60% of the total amino acid sequence of DT It is a non-toxic part that constitutes the hydroxylase. For example, Gill, D. M. and Dinius, L. L., J Biol. Chem., 246, 1485-1491 (1971), Gill ,DMand Pappenheimer,Jr.,AM.,J Biol.Che m., 246, 1492-1495 (1971), Collier, RJand K. andel, J., J Biol. Chem., 246, 1496-1503 (1971 ), and Drazin, R., Kandel, J., and Collier, R.J. ., J Biol. Chem., 246, 1504-1510 (1971) stomach.

[0029] The complete amino acid sequence of diphtheria toxin has been published. ,L.,Bjorn,M.J.,Horn,G.,Fong,D.,Buck,GA. ,Collier,RJand Kaplan,DA,Proc.Natl.A. See cad Sci.USA 80, 6853-6857 (1983). Specifically, DTFB contains amino acid residues 194 to 535 of DT.

[0030] CRM 197 The carrier protein is a single amino acid substitution in fragment A at residue 52. It is a variant of DT that has been rendered non-toxic. 197 and DT is fragment B The major T cell epitope is located at the DT amino acid sequence. It was found mainly in the B fragment. Bixler et al., Adv Exp Me d Biol. (1989) 251:175-80, Raju et al., Eur. J.Immunol.(1995)25:3207-3214, Diethelm-Ok ita et al., J Infect Dis (2000) 181:1001-9, and McCool et al., Infect. and Immun. 67 (Sep Please refer to pp. 4862-4869 in J. Appl. Chem. Soc. 1999.

[0031] The use of DTFB as described herein involves the ADP-ribosylation activity domain of diphtheria toxin. The use of DTFB is required to ensure that at least 90%, 95%, or 100% of the DNA is nucleotides, including deletions, substitutions, and additions. %, or 99% sequence identity. Examples of variants include cysteine DTFB(C8) is a deletion or mutation of the ADP-ribosylation activation domain. Diphtheria toxin in which the amino acid residues are deleted and cysteine ​​201 is removed or mutated. The use of DTFB also includes a fragment covering the sequence 265-450 of DT, which contains published T cell epitopes (Bixler et al., Adv Exp Med Biol.(1989)251:175-80, Raju et al.,E (See ur. J. Immunol. (1995) 25:3207-3214). DTFB may be in the form of a monomer, dimer, or oligomer. Any protein conjugate containing FB or a fragment (full-length DT or is CRM 197 ), hybrid proteins, or conjugated proteins The use of DTFB includes chemically modified DTFB or fragments (i.e., PEGylation, non-natural amino acid modifications).

[0032] In one embodiment, DTFB is purified by subsequent adsorption chromatography. Native DT or mutant CRM197 It is produced from the enzymatic digestion and reduction of Purified DTFB, with or without mutation at the DT C201 residue, was found to be either full-length native or C201 mutated DT or CRM 197 or the burr from which the A fragment was cleaved. It is believed that Capto™ Adhere and C can be similarly prepared from Capto™ Adhere. Multimodal resins and chromatographs commercially available as apto™ MMC Tris concentrations above 50 mM during the feed cycle were not sufficient to purify the cleaved native DTFB. It is particularly known to provide excellent modes of operation.

[0033] In one embodiment, the preparation of DTFB contains up to 10 mM DTT. The free cysteine ​​at position 201 allows disulfide bond formation between DTFB monomers. In such cases, nickel prevents dimerization caused by the conjugation reaction. However, the conjugation reaction proceeds in an otherwise identical manner. If DTT is not used, dimerized DTFB will react with the hydroxyl group in the presence of nickel. In the presence of Ps, it is conjugated to Ps and inhibits the synthesis of the conjugate by sequestering residual inhibitory cyanide. The degree of jugation can be improved.

[0034] Removal of free cysteines in DTFB (mutation of DT C201) is achieved by multimodal resin It is expected that the same behavior will be observed in the disulfide bond formation between free cysteines. Removal of free cysteines eliminates the need for DTT, since dimerization by ATP is not feasible. Increasing the Tris buffer concentration and the sodium chloride elution buffer concentration is expected. and improved recovery of DTFB proteins from Capto MMC chromatography resin. DTFB purification has been demonstrated to improve performance using other multimodal resins. It is expected that this will be possible.

[0035] In one embodiment, the DTFB is a recombinant DTFB with or without mutation of the DT C201 residue. The vector is efficiently expressed and subsequently purified by a variety of techniques known to those skilled in the art.

[0036] In certain embodiments of the present invention, CRM 197 is used as a carrier protein. RM 197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin. In an embodiment, it is a Corynebacterium tuberculosis strain grown in a casamino acid and yeast extract based medium. In another embodiment, the bacterium is isolated from a culture of Bacillus diphtheriae strain C7 (β197). , CRM 197 was recombinantly engineered according to the method described in U.S. Pat. No. 5,614,382. Typically, CRM 197 ultrafiltration, ammonium sulfate precipitation, and and ion exchange chromatography. CRM 197 is a registered trademark of Pfenex Expression Technology Pseudomonas spp. (Pfenex Inc., San Diego, CA) was used. Fluorescens (Pseudommonas fluorescens) It is manufactured.

[0037] DTFB and its variants are also available for proteins (peptides) and saccharides. Other suitable carrier proteins include DT (diphtheria toxoid), TT (tetanus toxoid) or TT fragment C, Cough toxoid, cholera toxoid (see, for example, International Patent Application Publication No. 2004 / 08325 1), Escherichia coli LT, Escherichia coli ST, and Schweizer Contains additional inactivating bacterial toxins such as exotoxin A from Domonas aeruginosa. Bacterial outer membrane proteins, porins, and transferrin-binding proteins such as OMPC , Pneumococcal surface protein A (PspA, International Patent Application Publication No. WO 02 / 091 998), pneumococcal adhesin protein (PsaA), group A or C5a peptidase from group B streptococcus, or Haemophilus influenzae Protein D, detoxified ply in some way, e.g., dPLY-GMBS (International Patent See Application Publication No. 04 / 081515) or dPLY-formol Pneumococcal pneumolysin (Kuo et al., 1995, Infect Immunol. n 63;2706-13), PhtA, PhtB, PhtD, PhtE, and Pht Protein fusions, such as PhtX (including PhtDE fusions, PhtBE fusions) See International Patent Application Publication No. 01 / 98334 and International Patent Application Publication No. WO 03 / 54007. Ovalbumin, keyhole limpet hemocyanin (KLH), Bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) , PorB (from Neisseria meningitidis), PD (Haemophilus influenzae Enza protein D (see, e.g., EP 0 594 610 B), or Synthetic peptides (see EP 0378881 and EP 0427347) and its immunologically functional equivalents, heat shock proteins (International Patent Application Publication No. See WO 93 / 17712 and WO 94 / 03208), pertussis tance Protein (International Patent Application Publication No. 98 / 58668 and European Patent No. EP 0471 177), cytokines, lymphokines, growth factors or hormones (see US Pat. No. 6,237,163). See International Patent Application Publication No. 91 / 01146), N19 protein (Baral See doi et al., 2004, Infect Immun 72:4884-7. Various pathogen-derived antigens, such as Multiple human samples from the NIH-10001410 ... artificial proteins containing CD4+ T cell epitopes, iron uptake proteins (international patent pending) See Application Publication No. 01 / 72337), Clostridium difficile (C. difficile toxin A or B (International Patent Publication No. WO 00 / 61761 , see issue 1), and flagellin (Ben-Yedidia et al., 1 998, Immunol Lett 64:9) It can be used as a carrier protein.

[0038] CRM 176 , CRM 228 , CRM 45 (Uchida et al., 1973, J Biol Chem 218:3838-3844), CRM9, CRM 45 , C.R. M 102 , CRM 103 , and CRM 107and Nicholls and Yo ule in Genetically Engineered Toxins,Ed: Other mutations described in Frankel, Maecel Dekker Inc., 1992 Glu-148 to Asp, Gln, or Ser and / or Ala to Gly 158 deletions or mutations, and U.S. Pat. No. 4,709,017 or U.S. Pat. Other mutations disclosed in 50,740, including at least one or more residues Lys 51 6, mutations of Lys 526, Phe 530 and / or Lys 534, U.S. Pat. No. 5,917,017 or other mutations disclosed in U.S. Pat. No. 6,455,673 or other DT mutations such as the fragments disclosed in U.S. Pat. No. 5,843,711. Such DT variants can be used as second carrier proteins. It can also be used to generate DTFB variants containing the B fragment containing the region.

[0039] In one embodiment, the present invention provides a method for the production of a medicament comprising administering to a subject a therapeutic agent conjugated to one or more carrier proteins. Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, and 6 D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15 B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B , 35F, and 38. Immunogenic compositions comprising an idioside-protein conjugate and a pharmaceutically acceptable carrier In one embodiment of the invention, the immunogenic composition comprises a CRM 197Individually Adjugated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5, 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, containing or essentially containing capsular polysaccharides from serotypes 42, 43, or 44 In one embodiment of the present invention, the CRM 197 is the only It is a carrier protein.

[0040] In one embodiment, the immunogenic composition comprises a second carrier protein (the first carrier protein). 1, 2, 3, and 4 conjugated to a protein (which differs from the protein by at least one amino acid) , 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 2 0, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 3 one selected from at least one of 3F, 34, 35A, 35B, 35F, and 38; Capsular polysaccharides from additional Streptococcus pneumoniae serotypes were further analyzed. Preferably, the saccharide from a particular serotype is attached to multiple carrier proteins. Not conjugated to

[0041] In one embodiment of the invention, the immunogenic composition of the invention comprises a second carrier protein. and further comprising capsular polysaccharide from at least one additional serotype conjugated thereto. In these embodiments, the immunogenic composition comprises a CRM 197 Not a second carrier tamper 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, individually conjugated to proteins 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39 , containing capsular polysaccharides from serotypes 40, 41, 42, 43, or 44, Consisting essentially of or consisting of.

[0042] In one embodiment of the invention, the immunogenic composition comprises capsular polysaccharides from the N serotype. wherein N is 2, 3, 4, 5, 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, and N The capsular polysaccharides from each of the serotypes are 197 The first protein is In another embodiment of the invention, one, two, three... or The capsular polysaccharide from the N-1 serotype is conjugated to a first protein carrier. The capsular polysaccharides from N-1, N-2, N-3...1 serotypes are CRM 197 is conjugated to a second protein carrier different from

[0043] In one particular embodiment of the present invention, the present invention provides a CRM 197 Serum conjugated to Type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F, and provides a 15-valent immunogenic composition comprising the same.

[0044] Capsular polysaccharide from Streptococcus pneumoniae is a standard known to those skilled in the art. For example, polysaccharides can be isolated from bacteria, Microfluidization is achieved by known methods, preferably using a homogenizer. To some extent, they can be sized by electrochemical or chemical hydrolysis (e.g., (See State Patents Nos. 497,524 and 497,525). In one embodiment, each Streptococcus pneumoniae strains corresponding to the polysaccharide serotypes were cultured on soy-based The individual polysaccharides are then separated by centrifugation, sedimentation, and ultrafiltration. The purified product is purified through standard steps, including filtration. See, for example, U.S. Patent Application Publication No. 2008 / 0029994. See U.S. Patent Nos. 0286838 and 5,847,112. The amide is used to reduce the viscosity and / or the subsequent cleavage of the conjugated product. The capsular polysaccharide may be sized to improve filterability. Serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9 V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C , 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 2 One or more of 7, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 It is prepared from a number of

[0045] The purified polysaccharides are chemically activated so that they can react with carrier proteins. Once activated, each capsular polysaccharide can be individually attached to a carrier tag. Conjugated to proteins to form glycoconjugates. Polysaccharide conjugates The bodies can be prepared by known coupling techniques.

[0046] In one embodiment, chemical activation of polysaccharides and subsequent attachment to carrier proteins Conjugation is described in U.S. Patent Nos. 4,365,170, 4,673,574, and This is accomplished by the means described in US Pat. Nos. 4,902,506 and 4,902,506. Pneumococcal polysaccharides are prepared by the addition of sodium periodate, potassium periodate, or periodate. reacted with a periodate-based oxidizing agent such as arginine to oxidize adjacent hydroxyl groups. This results in random oxidative cleavage, yielding reactive aldehyde groups.

[0047] Direct attachment of oxidized polysaccharides to primary amine groups (mainly lysine residues) on protein carriers The direct amination coupling can be achieved by reductive amination. For example, Conjugation is performed by combining activated polysaccharides with carrier proteins in the presence of nickel. This is done by reacting a mixture of The conjugation reaction can be carried out in aqueous solution or in an organic solvent such as DMSO. For example, U.S. Patent Application Publication No. 2015 / 0231270A1 and European Patent No. 04711 See U.S. Patent Application Publication No. 2011 / 0195086A1. At the end of the conjugation reaction, any unreacted aldehydes can be removed by cleavage with a solution such as sodium borohydride. The capping is achieved by the addition of a strong reducing agent.

[0048] In one embodiment, prior to formulation, each pneumococcal capsular polysaccharide antigen is Individually purified from Coccus pneumoniae and activated to form reactive aldehydes followed by reductive amination with sodium cyanoboride in the presence of nickel The nickel is covalently conjugated to a first or second carrier protein. Residual inhibitory cyanoacrylates from the sodium cyanoborohydride reducing agent used in reductive amination It forms a chelate ring with ammonium chloride.

[0049] In certain embodiments, the conjugation reaction is carried out by reductive amination, In this case, nickel is used for greater conjugation reaction efficiency, and free cyanide is used for Transition metals are used to aid in the removal of cyanides. It is known that the amino groups and formaldehyde in proteins can be cleaved with sodium cyanoborohydride. It is known to improve the reductive methylation of aldehydes (Gidley et al. , 1982, Biochem J 203:331-334, Jentoft et a. l., 1980, Anal Biochem. 106:186-190). By chelating the anhydride, the addition of nickel reduces the amount of tungsten in the conjugation. This increases protein consumption and leads to the formation of larger, potentially more immunogenic conjugates. Brings about formation.

[0050] Variation in free cyanide levels among commercially available sodium cyanoborohydride reagent lots is This results in inconsistent conjugation performance, resulting in variations in molecular mass and polysaccharide size. This may result in variable conjugate properties, including peptide-to-protein ratios. The addition of nickel to the conjugation reaction reduced the level of free cyanide, Thus improving the degree of lot-to-lot conjugate consistency.

[0051] In another embodiment, the conjugation method comprises the step of: Activation of polysaccharides with dibenzonium tetrafluoroborate (CDAP) was used to synthesize Activated saccharides can bind directly to amino groups on carrier proteins to form an anion ester. It can be closely coupled.

[0052] In another embodiment, the reactive homobifunctional or heterobifunctional group comprises a cyanate ester. Activation policers can be activated by reacting with one of several available modalities. For example, cystamine or cysteamine can be used to induce maleic anemia. Imido-activated carrier proteins (e.g., using GMBS) or haloacetylated carrier proteins Proteins (e.g., iodoacetimide (e.g., ethyl iodoacetimide HCl) or or N-succinimidyl bromoacetate or SIAB, or SIA, or SB thioether bond obtained after reaction with thiol (using AP) In another embodiment, the cyanate ester may be a hexasaccharide. reacted with benzodiamine or adipic acid dihydrazide (ADH) to produce amino derivatives Conjugated saccharides can be synthesized using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are then conjugated to free carboxy groups on the carrier protein. The gate body is disclosed in International Patent Application Publication Nos. 93 / 15760, 95 / 08348 and and WO 96 / 29094, and Chu et al., 1983, Inf ect.Immunity 40:245-256.

[0053] Other suitable conjugation methods include carbodiimides, hydrazides, active esters, Norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC , and TSTU. Many of these are described in International Patent Application Publication No. WO 98 / 42721. Conjugation is achieved by the reaction of the free hydroxyl groups of the saccharide with CDI. (Bethell et al., 1979, J. Biol. Chem. 254:257 2-4;Hearn et al.,1981,J.Chromatogr.218:5 09-18), followed by the carrier protein to form the carbamate bond. This chemical reaction can involve a carbonyl linker that can be formed by reaction with a substrate. reduction of the anomeric end of the carbohydrate to form a secondary hydroxyl group, followed by carbamyl Reaction of CDI with primary hydroxyls to form carboxylate intermediates and protein carriers The reaction consists of subsequent coupling to the amino group of another primary hydrochloride on the saccharide. Optional protection / deprotection of the xyl group may be required.

[0054] Following conjugation, the polysaccharide-protein conjugate is concentrated. Condensation / diafiltration, ultrafiltration, precipitation / elution, column chromatography and removing excess by one or more of any techniques known to those skilled in the art, including depth filtration. Removes conjugation reagents and residual free proteins and polysaccharides See, for example, U.S. Patent No. 6,146,902.

[0055] After the individual glycoconjugates are purified, they can be used to formulate the immunogenic compositions of the invention. These pneumococcal conjugates are prepared by different methods and are combined in a single It is bulk formulated into single dose formulations.

[0056] Pharmaceutical / vaccine compositions The present invention relates to the above polysaccharide having a pharmaceutically acceptable carrier and an adjuvant. comprising, consisting essentially of, or alternatively consisting of any of the serotype combinations Compositions, including pharmaceutical, immunogenic, and vaccine compositions, are further provided. So, the compositions are 2-13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 3 6, 37, 38, 39, 40, 41, 42, 43, or 44 different polysaccharides - comprising, consisting of or alternatively consisting of a protein conjugate, wherein each of the conjugates is a first carrier protein or a second carrier containing different capsular polysaccharides conjugated to proteins, where Leptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, and 6D 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23 A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 3 5F, and capsular polysaccharides from at least one of 38 are CRM 197 Choose from conjugated to a first carrier protein of choice, and and an adjuvant, together with a second carrier protein (at least one different from the first carrier protein). Serotypes 1, 2, 3, 4, 5, 6A, and 6B conjugated to ribozymes (which differ by one amino acid) , 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A , 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35 Additional Streptococcus pneumoniae strains selected from 35A, 35B, 35F, and 38 It may have the E serotype.

[0057] The formulation of the polysaccharide-protein conjugates of the present invention is well known in the art. This can be achieved using recognized methods. For example, 15 individual pneumococcal conjugates can be used. The gate body can be formulated with a physiologically acceptable vehicle to prepare a composition. Examples of such vehicles include water, buffered saline, polyols (e.g., glycerol, , propylene glycol, liquid polyethylene glycol) and dextrose solution Examples include, but are not limited to:

[0058] In another embodiment, the vaccine composition comprises an L-histidine buffer with sodium chloride. It is formulated in

[0059] As defined herein, an "adjuvant" is an agent that enhances the immunogenicity of the immunogenic compositions of the invention. When administered alone, immunoadjuvants can have adverse effects, e.g. or against weakly immunogenic antigens that induce no or weak antibody titers or cell-mediated immune responses. enhance the immune response to an antigen, increase antibody titers to an antigen, and / or achieve an individual's immune response. Therefore, adjuvants are often used to stimulate the immune response. and are well known to those skilled in the art. Suitable adjuvants for (1) Aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc. Alum, (2) For example, (a) Model 110Y Microfluidizer Submicrofluidizers such as uidics, Newton, MA 5% squalene, 0.5% Tween 80, and 0.5% Sp formulated into lon particles MF59 (International Patent Application No. 0900022002) containing an85 (which may contain various amounts of MTP-PE) (b) microfluidized to a submicron emulsion; or vortexed to produce emulsions with larger particle sizes. 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L 121, and SAF containing thr-MDP, (c) 2% squalene, 0.2% Tw een80, and the 3-O-deacylation described in U.S. Pat. No. 4,912,094. Monophospholipid A (MPL™), trehalose dimycolate (TDM), and cells wall skeleton (CWS), preferably MPL+CWS (Detox™) Ribi™ Adjuvant System (RAS) containing one or more bacterial cell wall components , (Corixa, Hamilton, MT), and (d) Montanide IS Oil-in-water emulsion formulations such as A (muramyl peptides (defined below) or bacterial with or without other specific immunostimulatory agents such as cell wall components), (3) Quil A or STIMULON™ QS-21 (Antigenic saponin adjuvants such as saponin adjuvants (e.g., U.S. Pat. No. 5,629,493; U.S. Pat. No. 5,629,493; and ... See US Pat. No. 5,057,540) can be used, or ISCOM (cholesterol Immunostimulatory agents formed by a combination of hydroxyl, saponin, phospholipids, and amphipathic proteins ISCOM complexes) and Iscomatrix™ (which have essentially the same structure as ISCOMs) particles produced therefrom, such as granules containing granules (which contain granules but no proteins), (4) Bacterial lipopolysaccharide, available from Corixa, U.S. Pat. No. 6,449,499; Aminoalkyl glucosamine phosphate compounds (AGPs) described in US Pat. No. 5,113,918 ), or a derivative or analog thereof; one such AGP is , 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy Oxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl] yl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD -glucopyranoside, which is also known as 529 (formerly known as RC529) (formerly known as acetaminophen), formulated in aqueous form or as a stable emulsion, (5) Synthesis of oligonucleotides containing one or more CpG motifs Polynucleotides (U.S. Patent No. 6,207,646), (6) Interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL- cytokines such as IL-6, IL-7, IL-12, IL-15, IL-18, etc., -ferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor Macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor ( TNF), costimulatory molecules such as B7-1 and B7-2, and (7) Complement, such as the trimer of complement component C3d Including, but not limited to:

[0060] In another embodiment, the adjuvant is a mixture of two, three or more of the above adjuvants. compounds, such as SBAS2 (also containing 3-deacylated monophosphoryl lipid A and QS21) It is an oil-in-water emulsion containing

[0061] Muramyl peptide is N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), and N-acetyl-normuramyl-L-alanine-2-(1'- 2'-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine This includes, but is not limited to, MTP-PE.

[0062] In one embodiment, the adjuvant is an aluminum salt. The vaccine can be an alum-precipitated or alum-adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harl ow, E. and D. Lane (1988; Antibodies: A Labora tory Manual Cold Spring Harbor Laboratories y) and Nicklas, W. (1992; Aluminum salts.Rese Arch in Immunology 143:489-493). Aluminum salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), and aluminum. Aluminum Hydrate, Aluminum Trihydrate, Alhydrogel®, Super rfos, Amphogel®, aluminum hydroxide (III), hydroxy Aluminum phosphate sulfate (aluminum phosphate adjuvant (APA)), amorphous aluminum alumina, alumina trihydrate, or aluminum trihydroxide do not have.

[0063] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is also known as aluminum chloride. Aluminum hydroxyphosphate is prepared by blending aluminum and sodium phosphate in a 1:1 volume ratio. After the blending process, the materials are subjected to high shear The product is then reduced in size in a mixer to achieve a monodisperse particle size distribution. Diafilter and steam sterilize.

[0064] In one embodiment, commercially available Al(OH)3 (e.g., from Denmark / Accurat e Chemical and Scientific Co., Westbury, N. 50-200g of Alhydrogel (registered trademark) or Superfos It is used to adsorb proteins at a ratio of protein / mg aluminum hydroxide. In another embodiment, protein adsorption is determined by the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with lower pIs are more likely to bind to the ATP than proteins with higher pIs. The aluminum salts adsorb to the strongly positively charged aluminum ions. Establish a reservoir of Ag that is slowly released over time, preventing nonspecific activation of macrophages and and complement activation and / or stimulate innate immune mechanisms (possibly through stimulation by uric acid). See, e.g., Lambrecht et al., 2009, Curr Opinion See Immunol 21:23.

[0065] The monovalent bulk aqueous conjugates are typically blended together and diluted. Once diluted, the batch is sterile filtered. Aluminum phosphate adjuvant is added at 8 μg / mL. Diluted to a target of 4 μg / mL for all serotypes except 6B concentration, and aseptically added to target a final aluminum concentration of 250 μg / mL The adjuvant formulation batch will be filled into vials or syringes. do.

[0066] In certain embodiments, the adjuvant comprises a CpG-containing nucleotide sequence, e.g., a CpG-containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG ODNs) In another embodiment, the adjuvant is ODN 1826, which is It can be obtained from Ley Pharmaceutical Group.

[0067] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "Nucleotide" and similar terms refer to a sequence of 6 to 50 nucleotides in length containing an unmethylated CpG moiety. It represents the nucleotide molecule of the nucleotide. For example, Wang et al., 2003, Va See, e.g., 21:4297. In another embodiment, any other term Art-accepted definitions are intended. A CpG-containing oligonucleotide is any Modified oligonucleotides using synthetic internucleoside linkages, modified bases and / or modified sugars Contains octide.

[0068] The use of CpG oligonucleotides is well known in the art, e.g. Sur et al., 1999, J Immunol. 162:6284-93, Verthelyi,2006,Methods Mol Med.127:139-5 8, and Yasuda et al., 2006, Crit Rev Ther Dr ug Carrier Syst.23:89-110.

[0069] Administration / Dosage The compositions and formulations of the present invention allow for the administration of vaccines via systemic or mucosal routes. to protect or treat humans susceptible to infectious diseases, such as pneumococcal infections, by In one embodiment, the present invention provides an immunologically effective amount of an immunizing agent of the present invention. Streptococcus pneumoniae capsular polyclonal antibody (SNP) containing an immunogenic composition for the treatment of Streptococcus pneumoniae. Methods for inducing an immune response to a saccharide conjugate are provided. In one embodiment, the present invention provides a method for administering to a human an immunologically effective amount of an immunogenic composition of the present invention. The present invention provides a method for vaccinating humans against pneumococcal infection, comprising administering a vaccine to the host.

[0070] The optimal amounts of components for a particular vaccine can be determined by standardized testing with observation of an appropriate immune response in subjects. For example, in another embodiment, the efficacy of the vaccine can be confirmed by human vaccination. Dosages for specific species are determined by extrapolation from animal studies to human data. In some cases, dosages are empirically determined. The data demonstrate that the vaccine is immunogenic.

[0071] An "effective amount" of the composition of the present invention is a dose that will prevent the growth of microorganisms, such as streptococci, during subsequent challenge. Induce antibodies that significantly reduce the infectious potential or severity of Caspian virus infection represents the dose required.

[0072] The method of the present invention is applicable to the treatment of invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute Otitis media, otitis media, and sinusitis) and other microorganisms, such as Streptococcus niger For the prevention and / or alleviation of the primary clinical syndrome caused by flumonea It can be used.

[0073] Administration of the compositions of the present invention may be via intramuscular, intraperitoneal, intradermal or subcutaneous routes, or orally. / may include one or more of injection via mucosal administration into the gastrointestinal, respiratory or genitourinary tract In one embodiment, intranasal administration is used to treat pneumonia or otitis media (pneumonia). Nasopharyngeal carriage of the bacterium can be prevented more effectively, thereby attenuating infection at its earliest stage ).

[0074] The amount of conjugate in each vaccine dose induced an immunoprotective response without significant adverse effects Such an amount may vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose is between 0.1 and 100 μg, particularly 0.1 to 10 μg, especially 1 to 5 μg of each polysaccharide. For example, each dose may be 100, 150, 200, 250, 300, 400, 500, or or 750 μg or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60 , 70, 80, 90, or 100 μg.

[0075] The optimal amounts of components for a particular vaccine can be determined by standardized testing with observation of an appropriate immune response in subjects. For example, in another embodiment, the efficacy of the vaccine can be confirmed by human vaccination. Dosages for specific species are determined by extrapolation from animal studies to human data. In certain circumstances, dosages are empirically determined.

[0076] 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, In yet another embodiment, the aluminum content is 1.2, 1.5, 2, 3, 5 mg or more. The dose of ammonium salt is per μg of recombinant protein.

[0077] In a specific embodiment of the present invention, the PCV15 vaccine is individually CRM 197 Conjugated to Gated serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, Sterile liquid preparations of 19F, 22F, 23F, and 33F pneumococcal capsular polysaccharides In one embodiment, each dose is 4 μg / mL, except for 6B, which is 8 μg / mL or 16 μg / mL. g / mL or 8 μg / mL of each saccharide, and approximately 64 μg / mL or 128 μg / mL CRM in g / mL 197 In one embodiment, each The 0.5 mL dose contains 2 μg of each saccharide, except for 6B, which contains 4 μg, and approximately 32 μg of CRM. 197 Carrier protein (e.g., 32 μg ± 5 μg, ± 3 μg, ± 2 μg, or ± 1 μg) g), 0.125 mg of elemental aluminum (0.5 mg of aluminum phosphate) Ajuba It is formulated to contain cereals containing cereals containing cereals containing glutathione, sodium chloride, and L-histidine buffer. The thorium concentration is approximately 150 mM (e.g., 150 mM ± 25 mM, ± 20 mM, ± 15 mM). M, ±10 mM, or ±5 mM), and about 20 mM (e.g., 20 mM ±5 mM, ± 2.5 mM, ±2 mM, ±1 mM, or ±0.5 mM) of L-histidine buffer. .

[0078] According to any of the methods of the present invention, in one embodiment, the subject is a human. In this context, the human subject may be an infant (under 1 year of age), a toddler (approximately 12-24 months), or a young child ( In other embodiments, the human subject is an elderly patient (>65 years old). The compositions of the invention are suitable for use in older children, adolescents, and adults (e.g., 18-45 years or 18-65 years). It is also suitable for use in

[0079] 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 vaccine is administered two, three, four or more times, sufficiently spaced apart. For example, the composition may be administered at 1, 2, 3, 4, 5, or 6 month intervals, or any combination thereof. Immunization schedules should follow those specified for pneumococcal vaccines. For example, invasive diseases caused by Streptococcus pneumoniae In contrast, the usual schedules for infants and toddlers are 2, 4, 6 and 12-15 Thus, in another embodiment, the composition is administered at 2, 4, 6, and 12-15 months of age. It is administered as a four-dose series at 1 month of age.

[0080] The compositions of the present invention contain one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion are In International Patent Application Publication No. 02 / 083855 and International Patent Application Publication No. WO 02 / 053761 Including those that have been identified.

[0081] formulation The compositions of the present invention can be administered parenterally, transmucosally, transdermally, intramuscularly, intravenously, or intradermally. by one or more methods known to those skilled in the art, such as intranasally, subcutaneously, or intraperitoneally. It may be administered to elephants and formulated accordingly.

[0082] In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous ... It is administered via intravenous, subcutaneous, or intramucosal injection into the respiratory tract. Includes liquids, etc.

[0083] The compositions of the present invention may be provided as single dose vials, multi-dose vials, or pre-filled. It may be formulated as a dispensing syringe.

[0084] In another embodiment, the compositions of the present invention are administered orally and therefore in a form suitable for oral administration. Solid oral formulations include tablets, capsules, and the like. Liquid oral preparations include liquids, suspensions, dispersions, etc. , emulsions, oils, etc.

[0085] 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. Aqueous carriers include saline and and buffered media, including water, alcoholic / aqueous solutions, emulsions, or suspensions. Examples of oils are those of animal, vegetable, or synthetic origin, e.g., peanut oil, soybean oil, olive oil, These include corn oil, sunflower oil, fish liver oil, other fish oils, or fats from milk or eggs.

[0086] Pharmaceutical compositions can be isotonic, hypotonic or hypertonic. or a pharmaceutical composition for injection that is essentially isotonic when it is administered. Therefore, for storage purposes, pharmaceutical compositions are preferably isotonic or If the pharmaceutical composition is hypertonic for storage, it may be administered It may be diluted to an isotonic solution before use.

[0087] 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 are NaCl, CaCl2, KCl, and MgCl2. Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol and glycerol.

[0088] It is also preferred that the at least one pharmaceutically acceptable excipient is a buffering agent. For some purposes, for example, when the pharmaceutical composition is for infusion or injection, the composition may be buffered. It is often desirable to include a buffering agent, which buffers the solution to 5-9, e.g., 6-8, etc. It can be buffered to a pH range of 4-10.

[0089] Buffers include, for example, Tris, acetate, glutamate, lactate, maleate, tartaric acid Salt, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, amber The buffer may be selected from the group consisting of acid salts, and triethanolamine buffers.

[0090] Buffering agents may be used, for example, in the preparation of parenteral preparations, particularly when the pharmaceutical preparation is for parenteral use. For example, the buffering agent may be selected from USP compatible buffers for acetic acid, benzoic acid, , gluconic acid, glycerin, and monobasic acids such as lactic acid, aconitic acid, adipic acid, Dibasic acids such as scorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid , polybasic acids such as citric acid and phosphoric acid, ammonia, diethanolamine, glycine, It may be selected from the group consisting of bases such as triethanolamine and Tris.

[0091] The parenteral vehicle (for subcutaneous, intravenous, intraarterial, or intramuscular injection) is a 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 surfactants and other pharmaceutically acceptable salts. Sterile liquids such as water and oil, with or without the addition of acceptable adjuvants. water, saline, aqueous dextrose and related sugar solutions, propylene glycol or Glycols such as polyethylene glycol, polysorbate 80 (PS-80), poly Sorbate 20 (PS-20) and Poloxamer 188 (P188) are particularly suitable for injections. Examples of oils are those of animal, vegetable, or synthetic origin, e.g., pectin, glycerin, glycerol, sorbitan, sorbitan-based ... - Nut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other fish oils, or milk or egg It is a lipid from

[0092] The formulations of the present invention may also contain a surfactant. Preferred surfactants are polyoxyethylene isosorbitan ester surfactants (commonly referred to as Tweens), especially PS-20 and PS-80; a DOWFAX™ product name such as a linear EO / PO block copolymer Ethylene oxide (EO), propylene oxide (PO), and / or or copolymers of butylene oxide (BO); octoxynol, which is a repeating ethoxylated The number of oxy(oxy-1,2-ethanediyl) groups can vary, and octoxynol-9 (Tr Iton X-100, or trioctylphenoxypolyethoxyethanol) are particularly Interesting; (octylphenoxy)polyethoxyethanol (IGEPAL CA-63 0 / NP-40); phospholipids such as phosphatidylcholine (lecithin); Tergito Nonylphenol ethoxylates such as the NP series; triethylene glycol Lauryl, cetyl, stearyl, and methyl lauryl ether (Brij30) Polyoxyethylene derived from oleyl alcohol (known as Brij surfactant) fatty ethers; and sorbitan trioleate (Span 85) and monolaurin Sorbitan esters such as sorbitan acid (commonly known as SPAN) , but not limited to these.

[0093] Mixtures of surfactants may be used, for example, a PS-80 / Span 85 mixture. Polyoxyethylene sorbitan monooleate (PS-80) Tanester and t-octylphenoxypolyethoxyethanol (Triton X- Combinations with octoxynol such as 100 are also suitable. Another useful combination is laureth. and polyoxyethylene sorbitan ester and / or octoxynol. nothing.

[0094] Preferred amounts of surfactant are as follows: polyoxyethylene sorbitan esters (PS-80, etc.) 0.01 to 1 w / v%, especially about 0.1 w / v%, octyl or Nonylphenoxypolyoxyethanol (Triton X-100, or Trito Other detergents in the N series) 0.001 to 0.1 w / v%, especially 0.005 ~0.02 w / v%, polyoxyethylene ether (such as laureth 9) 0.1~20 w / v%, preferably 0.1 to 10 w / v%, in particular 0.1 to 1 w / v% or about 0.5 w / v %.

[0095] In one embodiment, the composition contains 250 μg / mL of APA (aluminum phosphate adjuvant). Bant) together with L-histidine (20 mM), saline (150 mM) at pH 5.8, and Consists essentially of 0.2 w / v% PS-20. PS-20 was used during simulated manufacturing and primary packaging. When PS-20 is present in the formulation, it controls aggregation during transport using a 0.00 The process may contain L-listidine, sodium chloride, and and a blend of up to 44 serotypes in PS-20, and then combining this blend The materials were combined with APA and sodium chloride with or without antimicrobial preservatives. It consists of:

[0096] As demonstrated herein, the choice of surfactant can be varied for different drug products and drug substances. For multivalent vaccines containing 15 or more serotypes, the vaccine may need to be optimized. , PS-20 and P188 are preferred. The choice of chemistry is believed to be an important factor influencing the stability of the formulation. In particular, in multivalent compositions prepared in aqueous or DMSO solvents, as exemplified below: The combined pneumococcal polysaccharide-protein conjugates are used in formulations They show significant differences in stability depending on the particular surfactant system used. In particular, one or more polysaccharide-protein conjugates may be used in a solvent such as DMSO. When prepared in aprotic solvents, polysorbate 20 alone or in combination with polyols Improved stability was observed with combined poloxamer 188.

[0097] The present invention relates to reduced hydroxybenzoates, some of which are prepared under aqueous conditions and others under DMSO conditions. Polysaccharide-protein conjugates prepared using reactive amination Combination of polysorbate 20 or poloxamer 188 with polyols in formulations with The use of the immunogenic composition helps control manufacturing and transportation stress-induced physicochemical instability. This was due in part to the discovery that these surfactants offer unexpectedly superior properties compared to other surfactants and stabilizers. The exact mechanism by which specific detergents protect biotherapeutics is unclear. Possible stabilization mechanisms include preferential hydration, preferential Prior exclusion, competition for the air / liquid interface between the biotherapeutic and the surface, surface tension, and / or aggregation Detergents with biotherapeutics to mask hydrophobic patches that act as aggregation seeds The present invention relates to immunological assays comprising polysaccharide-protein conjugates. The present invention provides a method for improving the stability of an active ingredient composition and inhibiting particulate formation (e.g., aggregation, precipitation). The formulations of the present invention comprise poloxamer 188 and Complex biotherapeutic options are available for previously used surfactants, including polysorbate 80. These compounds are believed to offer significant advantages in controlling manufacturing, transport, and handling-induced aggregation of therapeutic agents. It is given.

[0098] Protein components in polysaccharide-protein conjugates play an important role in aggregation This is because the same serotype composition but different conjugation Different aggregation phenomena of drug products using conjugates of hydroxypropyl methylcellulose and hydroxypropyl methylcellulose have been demonstrated. The aprotic solvent used in the preparation of the polysaccharide conjugates is a protein may have different structures and exhibit different propensities to aggregate in the presence of APA adjuvant. If carrier protein is used, the weight percentage can be calculated.

[0099] Thus, in one embodiment of the present invention, the present invention provides a method for producing a polysaccharide comprising: (i) one or more polysaccharides; (ii) a pH in the range of 5.0 to 7.5; (ii) an aluminum salt; and (iv) a) polysorbate 20 and and (b) poloxamers having molecular weights in the range of 1100 Da to 17,400 Da; a polyol selected from propylene glycol and polyethylene glycol 400; In some aspects of this embodiment, a surfactant system selected from one or more The polysaccharide-protein conjugate or conjugates may be dissolved in an aprotic solvent such as DMSO. It is prepared in a solvent that accounts for approximately 10-100%, 24%-100%, or The range of 24-80% is prepared in aprotic solvents such as DMSO, and the conjugated It is possible.

[0100] In one embodiment, the surfactant system is a commercially available surfactant commonly referred to as Tween® 20. Polysorbate 20 (IUPAC name: Polyoxyethylene (20)), a commercially available surfactant In one embodiment, the formulations of the present invention comprise a sorbitan monolaurate (PS-20). The final concentration of polysorbate 20 was 0.001 w / v% to 10 w / v%, 0.025 w / The range is between 0.025w / v% and 0.3w / v%. The surfactant system comprising Sorbate 20 may further comprise a polyol. propylene glycol and polyethylene glycol. Ethylene glycol or propylene glycol is 6 w / v% to 20 w / v% final In one embodiment, the polyethylene glycol is polyethylene glycol 400 is.

[0101] In one embodiment, the surfactant system comprises a surfactant having a molecular weight in the range of 1100 Da to 17,400 Da. Poloxamer having the formula α-methylpropyl methylcellulose, propylene glycol, and polyethylene glycol 400 and a polyol selected from the group consisting of:

[0102] Poloxamers are made up of two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). It consists of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by Poloxamers are nonionic triblock copolymers that are marketed under the trade name Pluronic (registered trademark). The length of the polymer block can be customized. Therefore, there are many different poloxamers with slightly different properties. These copolymers are generally designated by the letter "P" (for poloxamer). The name is given with a three-digit number after the first two digits x 100, which means polyoxypropylene. Indicates the approximate molecular mass of the core, and the last digit x 10 indicates the polyoxyethylene content. (For example, P407 = polyoxypropylene molecular mass of 4,000 g / mol and 70 % polyoxyethylene content). In terms of trade names, the coding of these copolymers is used to define their physical form at room temperature. Start with the first letter (L=liquid, P=paste, F=flake (solid)), then 2 or Three digits follow. The first digit of the numerical display (the second digit of the three-digit number) multiplied by 300 is the hydrophobicity indicates the approximate molecular weight of the substance, and the last digit x 10 indicates the polyoxyethylene content ( For example, L61 = 1,800 g / mol of polyoxypropylene molecular mass and 10% polyoxypropylene Pluronic® with trioxyethylene content. U.S. Patent No. 3,744,444 Please refer to issue 0,421.

[0103] An example of a poloxamer has the general formula: HO(CHO) a (C3H6O) b (C2H4O) a H, where the a and b blocks have the following values: [Table 1]

[0104] As used herein, the molecular weight unit is Dalton (Da) or g / mol .

[0105] Regarding formulation, poloxamers are available in the following ranges: 1100 Da to 17,400 Da, 7,500 Da to Generally have a molecular weight of 15,000 Da, or in the range of 7,500 Da to 10,000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulation of the present invention is 0.001 to 5 w / v%, or 0.0 Poloxamer-containing surfactant systems also contain polyols. In one embodiment, the polyol is propylene glycol and has 1 to 2 In one embodiment, the polyol is polyethylene glycol 4. 00, with a final concentration of 1 to 20 w / v%.

[0106] Suitable polyols for the formulation include polymeric polyols, especially propylene glycol and polyols. polyethylene glycol, polyethylene glycol monomethyl ether, The polyether diols include, but are not limited to, propylene glycol, from about 425 to about 2 Available in a range of monomer molecular weights from 700 to 700. Ethylene glycol monomethyl ether also has a molecular weight range of about 200 to about 35,000. Available in PEG200, PEG300, PEG400, PEG1000, PE GMME550, PEGMME600, PEGMME2000, PEGMME3350, and PEGMME4000. The polyol is polyethylene glycol 400. Final concentration of polyol in the formulation of the present invention can be 1 to 20 w / v% or 6 to 20 w / v%.

[0107] The formulation also contains a pH buffered saline solution. Buffering agents include, for example, Tris, acetate, glutamine, and the like. Salt, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate , L-histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl) -1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), acid), MES (2-(N-morpholino)ethanesulfonic acid), and triethanolamine The buffer may be selected from the group consisting of 4 to 10, 5.2 to 7.5, or amine buffers. can buffer the solution to a pH in the range of 5.8 to 7.0. Buffers include phosphate, succinate, L-histidine, MES, MOPS, HEPES, and acetic acid. The buffering agent is selected from the group consisting of, for example, a phosphate, or a citrate, especially in pharmaceutical formulations. If the formulation is for parenteral use, then the formulation must be prepared in accordance with USP compatible buffers for parenteral use. The concentration of the buffer may range from 1 mM to 50 mM or from 5 mM to 50 mM. In one embodiment, the buffer contains L-histidine at a final concentration of 5 mM to 50 mM, or is succinate at a final concentration of 1 mM to 10 mM. In one embodiment, L-histidine is The final concentration is 20 mM ± 2 mM.

[0108] Although saline (i.e., a solution containing NaCl) is preferred, other salts suitable for the formulation include: Including, but not limited to, CaCl2, KCl and MgCl2, and combinations thereof Sucrose, trehalose, mannitol, sorbitol, and glycerol Non-ionic isotonic agents, including but not limited to, may be used in place of salts. The range includes, but is not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one embodiment, the saline solution is NaCl and is present at a concentration of 20 mM to 170 mM. That's fine.

[0109] In a preferred embodiment, the formulation comprises an L-histidine buffer with sodium chloride. .

[0110] In certain embodiments of the formulations described herein, the polysaccharide-protein conjugate The carrier protein comprises one or more pneumococcal polysaccharides conjugated to a carrier protein. Carrier proteins include CRM 197 , diphtheria toxin fragment B (DT FB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, Escherichia coli LT , Escherichia coli ST, exotoxin A from Pseudomonas aeruginosa, and its In some embodiments, one or more polysaccharide-protein combinations may be selected. The protein conjugate is conjugated to DTFB. All of the peptide-protein conjugates are prepared using aqueous chemistry. As a result, polysaccharide-protein conjugate formulations have been used in CRM 197 Polypeptide Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, and 18 conjugated to Streptococcus neutrophils from C, 19A, 19F, 22F, 23F, and 33F A 15-valent pneumococcal conjugate (15vPnC) consisting essentially of Monnier polysaccharides In another embodiment, the polysaccharide-protein conjugate may be formulated as a One or more of the compounds are prepared using DMSO chemistry. Kalide-protein conjugate preparations are effective against serotypes 6A, 6B, 7F, 18C, and 19A. Polysaccharide-protein conjugates from 19F and 23F were synthesized using DMS Prepared using the O chemical reaction, serotypes 1, 3, 4, 5, 9V, 14, 22F, and 3 Polysaccharide-protein conjugates from 3F were prepared using aqueous chemistry. The formulation may be a 15-valent pneumococcal conjugate (15vPnC) formulation.

[0111] In another embodiment, the pharmaceutical composition is delivered in a sustained release system. For example, the agent can be administered intravenously. , transdermal patch, liposomes, or other modes of administration. In, for example, polymeric materials in microspheres or implants are used.

[0112] The compositions of the present invention contain one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion are In International Patent Application Publication No. 02 / 083855 and International Patent Application Publication No. WO 02 / 053761 Including those that have been identified.

[0113] 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 encompass within the scope of the invention as defined in the appended claims. Various changes and modifications may be made therein by those skilled in the art without departing from the spirit or scope of the present invention. Please understand that.

[0114] The following examples illustrate, but do not limit, the present invention.

[0115] [Example] Example 1: Preparation of DTFB carrier protein Use of multimodal anion exchange chromatography for DTFB preparation As previously described (see International Patent Application Publication No. WO 2012 / 173876 A1 Pseudomonas fluorescens Purified CRM obtained by expression in 197 50 mM Tris, pH 8 0.0 at about 22°C for about 1 hour at a molar ratio of 1:500 tyrosine to CRM 197 Using The fragments were digested with recombinant trypsin, followed by dithiothreitol in 50 mM Tris, pH 8. (DTT) was added to a final concentration of 5 mM for 30 min at approximately 22°C to induce proteolytic cleavage. CRM 197 The disulfide bond between the A and B fragments of was reduced.

[0116] The digestion reaction was then transferred to a multimodal anion exchanger equilibrated with 50 mM Tris, pH 8. A cation exchange chromatography column (Capto™ Adhere, GE Healthcare) was used. The column was washed with 50 mM Tris, pH 8, and DTFB-generated eluate. The material was dissolved in a gradient of 0.45M to 0.65M sodium chloride in 50mM Tris, pH 8. The product was filtered through a 5 kDa nominal molecular weight cut-off (NMWCO) tangential flow ultrafiltration membrane. The filtrate was concentrated and diafiltered against 10 mM potassium phosphate, pH 8. The retentate containing the DTFB product was 0.2 micron filtered and stored at 2-8°C. Product concentration was determined by absorbance at 280 nm, and purity was confirmed by SDS-PA under non-reducing conditions. Evaluated by GE.

[0117] The results show that the multimodal anion exchange chromatography eluent has a small amount of dimer. Figure 1A shows that the sample contained relatively pure DTFB, which exists primarily as a monomer. See, Dimer formation was attributed to disulfide bond formation between DTFB monomers. As exemplified in the following section, DTT is used in cloning to minimize the possibility of dimer formation. It has been used in chromatography and ultrafiltration steps.

[0118] Use of multimodal cation exchange chromatography for DTFB preparation Due to the presence of DTFB dimers, alternative purification methods were investigated. Purified CRM obtained by expression in Solanum fluorescens 197 300 mM Tris, Trypsin was diluted to a protein concentration of approximately 1 mg / mL using PBS, pH 7.5. 3250 Trypsin vs CRM 197 The molar ratio was added to the protein solution. The mixture was incubated at room temperature for approximately 20 hours. DTT was added to a final concentration of 10 mM DTT to remove proteolytically cleaved CRM. 197 The disulfide bond between the two fragments was reduced to separate the A and B fragments.

[0119] After approximately 75 minutes, the reduced protein solution was transferred to a multimodal cation exchange chromatography column. The column was loaded onto a Capto™ MMC column (GE Healthcare). Before loading the protein solution, add 300 mM Tris, 10 mM DTT, pH 7 The column was equilibrated with 0.5 at 2-8°C. After loading, the column was rehydrated with 200 mM sodium chloride and and washed with 300 mM Tris, pH 7.5 containing 10 mM DTT at 2-8°C. The product was then diluted with 1 M sodium chloride in 300 mM Tris, pH 8.5 at 2-8°C. Approximately 0.002 w / v% of PS-20 was eluted through a 5 kDa NMWCO tangential flow ultrafiltration membrane. After concentration, additional PS-20 was added to the batch prior to concentration at 2-8°C using 0. PS-20 was added to the batch to a concentration of 0.02 w / v% and the batch was diluted with 100 mM potassium phosphate. The batch was diafiltered against 5 kDa sodium, 10 mM DTT, pH 8. Further concentration was achieved using tangential flow filtration with a membrane. The final retentate was filtered through a 0.2 micron filter. The samples were filtered and then frozen or stored at 2-8°C before conjugation.

[0120] DTFB samples were analyzed by SDS-PAGE under reducing conditions (FIGS. 1B and 1C). Concentration analysis of the gel showed that the final bulk intermediate (FBI) was >98% pure after 0.2 micron filtration. Indicates that the degree is

[0121] The final bulk intermediate (FBI) shown in Figure 1C was analyzed by liquid chromatography with mass spectrometry. The intact protein mass was determined by LC-MS analysis. Analysis was performed on samples after reduction with DTT. Deconvolution of raw data from the main peak The solution yielded a measured mass of 37,194.2 Da. This mass measurement was confirmed by the DTF This matches the theoretical mass of 37,194.4 Da for B, confirming the predicted amino acid sequence. did.

[0122] Peptide maps were analyzed using trypsin, endoproteinase Asp-N, and endoproteinase The samples were obtained from a combination of enzyme Glu-C digestion in the presence of 6 M guanidine-HCl. The reaction mixture was subjected to reductive alkylation with iodoacetamide and incubated at 37°C for approximately 16-17 min with each enzyme separately. The digestion was quenched by the addition of formic acid. The peptides were separated and analyzed by LC-MS. Combinations of peptides identified by separate digests were used to analyze the amino acids. The coverage of the amino acid sequence was approximately 98%. The sequence matched.

[0123] An alternative multimodal cation exchange chromatography process for DTFB purification Purified CRM obtained by expression in Pseudomonas fluorescens as described above 197 Approximately 5mg / mL protein concentration using 300mM Tris, pH 7.5 Trypsin was diluted to approximately 1:3000 trypsin to CRM. 197 Using mole ratios The solution was incubated at about 22°C for about 15 to 20 hours. Add DTT in 300 mM Tris, pH 7.5 to a final concentration of ≥ 10 mM DTT. Addition of proteolytically cleaved CRM 197 Reduce the disulfide bond between A The A fragment and the B fragment were separated.

[0124] Reduced protein solution with approximately 25 g of protein per L of resin and multimodal cations Exchange chromatography column (Capto™ MMC, GE Healthcare e) The column was loaded with 300 mM Tris, The column was equilibrated with 10 mM DTT, pH 7.5 at approximately 22°C. After loading, the column was 300 mM Tris, pH 7 containing 0 mM sodium chloride and 10 mM DTT The product was then washed with 1 M NaCl in 300 mM Tris, pH 8.5 at approximately 22°C. Elution was performed with sodium at 22°C. The DTFB product was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The extract can be concentrated and 0.2 micron filtered as above.

[0125] DTFB samples from the multimodal cation exchange process were subjected to SDS-PA under reducing conditions. The gel was analyzed by GE (Figure 1D). This shows that the DTFB protein product eluted from the gel is highly purified.

[0126] Effect of buffer pH, ionic strength, and PS-20 interface on DTFB stability during ultrafiltration Effect of surfactant concentration Buffer pH, ionic strength, and PS-20 surfactant studies were performed to determine the DTFB ultrafiltration rate. We addressed the protein particle formation observed during the over-step development. 100 at pH 7, 7.5, or pH 8 with 0 or 500 mM sodium chloride The pH and sodium chloride were measured using differential scanning calorimetry. The stability (melting temperature, Tm) of DTFB solutions as a function of sodium concentration was evaluated. As shown, increasing the pH from 7 to 8 increased the DTFB Tm by approximately 3°C. Sodium chloride ranging from 0 mM to 10 mM sodium chloride significantly affected Tm at the pH values ​​investigated. was not given. [Table 2]

[0127] In a separate study, purified DTFB was diluted with 0, 150, 500, and 1000 mM sodium chloride. pH 6.0, pH 7.0, and pH 8 in 100 mM potassium phosphate with sodium The solution was kept at room temperature overnight and then centrifuged to remove the precipitated protein. The supernatant was analyzed by size exclusion chromatography with UV280 absorbance detection. The supernatant protein concentration in this study was 100%. was not affected by the solution pH at 0 and 150 mM sodium chloride. However, at higher sodium chloride concentrations (500 and 1000 mM), the results were mild. As the buffer pH was decreased from pH 7.0 or 8.5 to 6.0, the supernatant protein concentration , which indicates a decrease in DTFB stability.

[0128] The effect of PS-20 concentration on DTFB stability was investigated using 50 and 100 mM potassium phosphate. in a pH 8 solution and 50 mM potassium phosphate, 150 mM sodium chloride, The study was carried out by adding increasing amounts of PS-20 to a DTFB solution at pH 8. The supernatant was analyzed by UV280 absorbance detection. The samples were assayed for protein concentration by size exclusion chromatography (Figure 3). Significant vortex-induced protein loss was observed in samples without PS-20. The recovery of DTFB was significantly improved in samples containing ≥ 0.01 w / v% PS-20. Ta.

[0129] Example 2: Preparation of Streptococcus pneumoniae capsular polysaccharide Methods for culturing pneumococci are known in the art, see, e.g., Chase, 1967 ,Methods of lmmunology and immunochemist See, e.g., 1:52. Methods for preparing pneumococcal capsular polysaccharides are also described. It is known in the art. See, for example, European Patent No. 0497524. Subtype isolates were collected from the American Type Culture Collection (Manassas, Vt.) A) The bacterium is an encapsulated, non-motile strain that is alpha-hemolytic on blood agar. It has been identified as a Gram-positive, lancet-shaped diplococci. Subtypes are determined by specific antisera. For example, U.S. Patent No. 5,847, Please refer to issue 112.

[0130] Freeze cell banks representing each of the Streptococcus pneumoniae serotypes of interest. in a sealed vial at Merck Culture Collection (Rahway, NH). The thawed seed culture was obtained from the company (J). The culture was transferred to a seed fermentor containing sterilized growth medium. The entire volume of the seed fermentor was filled with a production flask containing pre-sterilized growth medium. The production fermentation was the final cell growth stage of the process. Temperature, pH, and The stirring speed was controlled.

[0131] The fermentation process was terminated through the addition of an inactivating agent. After inactivation, the batch was inactivated. The cells were then transferred to a tank where they were kept at controlled temperature and agitation. The cell debris was removed by centrifugation and filtration. The batch was then ultrafiltered and diafiltered using a combination of ultrafiltration and diafiltration. was subjected to a solvent-based fractionation to remove impurities and recover the polysaccharides.

[0132] Example 3: Polymerization of DTFB Carrier Proteins Using Reductive Amination in Aqueous Solution Conjugations of Caride Serotype 3-DTFB (ST3-DTFB) conjugate for mouse immunogenicity studies Body preparation Purified serotype 3 polysaccharide obtained as described in Example 2 was dissolved in water. The reduction in Ps size to an average molecular weight of 200 kDa was observed by probing the sample while it was cooled in ice. The sonicated samples were filtered at 0.2 microns and then filtered for 2-8 minutes. The polysaccharide solution was filtered through a 30 kDa NMWCO tangential flow filtration membrane. The extract was concentrated by ear filtration.

[0133] Conjugation of polysaccharides using sodium metaperiodate oxidation (Anderson et al., 1986, J Immunol. 13 7:1181-1186 and U.S. Patent Application Publication No. 20110195086. (I want to use this). 100 mM sodium metaperiodate solution is dissolved in 50 mM sodium acetate. The samples were incubated at 19-25°C for 14-18 hours protected from light. Ethylene glycol (100:1 molar excess relative to the polysaccharide repeating unit) was mixed. Add the remaining sodium metaperiodate and mix for another 16 to 18 hours at 19 to 25°C. The resulting solution was diluted with 10 volumes of water and diluted with 10 volumes of water. The oxidized polysaccharide solution was stored in aliquots at -70°C.

[0134] Periodate-oxidized polysaccharides were added at a ratio of 0.6:1 polysaccharide to protein mass. In the ratio, the hydroxyl groups were analyzed using multimodal anion exchange chromatography as described in Example 1. The solution was mixed with DTFB prepared as described above. Potassium phosphate, pH 6.4, and nickel chloride were added to a final concentration of 145 mM and 2.2 mM, respectively. 1 equivalent of sodium cyanoborohydride was added. The reaction was protected from light and heated at 2-8°C. The test was conducted for 120 hours.

[0135] The mixture was then incubated at 2-8°C for a total of 14-18 hours in two changes of 25 mM potassium phosphate buffer. The insoluble material was dialyzed against sodium buffer, pH 6.4, 0.3 M sodium chloride. The conjugates were removed by brief centrifugation and purified by size exclusion chromatography ( The tissue was polished by SEC (Separate Section) to reduce free polysaccharides and proteins. The polished conjugate was concentrated using a 30 kD NMWCO centrifugal concentrator.

[0136] Preparation of serotype 3-DTFB conjugates for immunogenicity studies in pup rhesus monkeys Purified serotype 3 pneumococcal capsular polysaccharide powder was dissolved in water and filtered through a 0.45 micron filter. The batch was homogenized to reduce the molecular mass of the Ps and filtered to 0.22 microns. The size of Streptococcus pneumoniae polysaccharide before conjugation The reduction in size is due to the use of polysaccharides with more specific, reproducible, and manageable physical properties. has been previously described as a means to produce As described by Marburg et al., Saccharide size reduction increases solubility and filterability and decreases polydispersity and viscosity. This reduces the conjugation consistency and ease of conjugation. It is known that homogenization improves the The reduction in polysaccharide size of the serotype 19F polysaccharide from S. humoniae is as previously described (Lander et al., 2000, Biotechnol. Frog. 2000, 16, 80-85). The size-reduced polysaccharides were then concentrated. and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Ta.

[0137] Then, 50 mM sodium acetate was added to form reactive aldehydes on the polysaccharide. Addition of 100 mM sodium metaperiodate solution to form polysaccharides Activation was initiated. The batches were incubated at about 22°C for about 12 hours. The batches were incubated for ≤8 hours. 10 mM potassium phosphate, p using a 10 kDa NMWCO tangential flow ultrafiltration membrane at 37 °C. The product-rich retentate was concentrated by diafiltration against H6.4.

[0138] The activated polysaccharide solution was prepared by blending water and 1.5 M potassium phosphate, pH 7.0. The purified DTFB was filtered at 0.2 microns and then diluted with 1.3:1 polysaccharide extract. The solution was then combined with the buffer-adjusted polysaccharide solution in a weight-to-protein ratio. Nickel chloride was prepared from a 100 mM nickel chloride stock solution. The batch was then added to a final concentration of approximately 2 mM using sodium cyanoborohydride. The batch was heated to about 10°C. The reaction was carried out at RT for approximately 120 hours to maximize the consumption of polysaccharides and proteins.

[0139] After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L. The mixture was cooled to 2-8°C, filtered through a 1.2 micron filter, and passed through a 100 kDa NMWCO tangential flow ultrafilter. Using a filter membrane, diaphragm the cells against 100 mM potassium phosphate, pH 7.0, at 2-8°C. The batch recovered in the retentate was then filtered to obtain approximately 2.0 g of polysaccharide. The solution was diluted to 0.5 mL / L and the pH was adjusted by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mole per mole of polysaccharide repeating unit) was added. 1.5 M potassium phosphate, pH 6.0 was then added.

[0140] The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. For 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at ~8°C The retentate was filtered through 0.2 microns and an additional 150 mM salt was added. 1.0 g / L polysaccharide was prepared using 10 mM L-histidine in sodium chloride, pH 7.0. The chloride concentration was adjusted. The batch was divided into aliquots and frozen at ≦−60°C.

[0141] Example 4: CRM using reductive amination in aqueous solution 197 serotypes 1, 3, and 4 , 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and Conjugation of 33F and 33F Different serotype polysaccharides were purified using a common process flow. 197 Individually conjugated to carrier proteins. Polysaccharides are dissolved and reduced in size. The purified CRM was then purified by ultrafiltration. 197 of NiCl2 (2 mM) in the reaction mixture was used to conjugate the activated polysaccharide. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Several process parameters within each step, such as pH, temperature, concentration, and time, were The data were controlled to serotype-specific values ​​in the following sections.

[0142] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and used for all sera except for serotype 19A. The types were filtered at 0.45 microns. Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14 , 19A, 19F, 22F, 23F, and 33F were homogenized to obtain polysaccharides. The molecular mass of the serotype 18C was reduced by homogenization at ≥90°C or acidification. Serotype 19A was found to be sizable due to its relatively low starting size. The size was not reduced due to the homogenization pressure and homogenizer. The number of passes was controlled to a serotype-specific target (150-1000 bar; 4-7 passes). The size-reduced polysaccharide was filtered to achieve a serotype-specific molecular mass. filtered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane and then concentrated. A diafiltered 5 kDa NMWCO membrane was used for acid hydrolyzed serotype 18C.

[0143] The polysaccharide solution was then diluted with sodium acetate buffer at serotype-specific temperatures ( The temperature was adjusted to 4-22°C and pH to 4-5 to increase the size of the polysaccharides by activation. For all serotypes (except serotype 4), the polysaccharide activation The reaction was initiated by the addition of 100 mM sodium metaperiodate solution. The amount of sodium phosphate is serotype specific and is approximately 1 mole of polysaccharide repeating unit. Sodium metaperiodate ranged from 0.1 to 0.5 molar. The serotype-specific charge of the ATP is intended to achieve the desired level of polysaccharide activation. (moles of aldehyde per mole of polysaccharide repeating unit). The batch was then incubated at approximately 50°C and pH 4.1 before adding sodium metaperiodate. The polysaccharide was partially deketalized by bating.

[0144] For all serotypes except serotypes 5 and 7F, the activation product was determined to be 10 kDa NM Dilute the solution in 10 mM potassium phosphate, pH 6.4 using a WCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for acid hydrolysis of serotype 18C. Types 5 and 7F were diafiltered against 10 mM sodium acetate. Ultrafiltration for the mold was carried out at 2-8°C.

[0145] CRM 197 Polysaccharide conjugation to Oxidized polysaccharide solution, depending on serotype, is prepared by adding water and 1.5 M potassium phosphate, p The selected buffer pH was adjusted for the conjugation reaction. The purpose of this study was to improve the stability of activated polysaccharides during the reaction. As such (see International Patent Application Publication No. 2012 / 173876A1), Purified CRM obtained through expression in Solanum fluorescens 197 0.2 microns and polysaccharide to CRM ranging from 0.4 to 1.0 w / w depending on serotype. 197 quality The resulting polysaccharide was combined with a buffered polysaccharide solution in a mass ratio of 0.01 to 0.01. Resaccharides vs. CRM 197 The ratio of polysaccharides and lysine was chosen to be controlled. The phosphate concentration is serotype-specific and ranges from 3.6 to 10.0 g / L and The conjugates that produced serotype-specific polysaccharide concentrations ranged from 100 to 150 mM. The size of the gate bodies was selected to control the size of the gate bodies. The solution was then filtered to 0.2 microns. Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution. Sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added Conjugation was allowed to proceed for a serotype-specific period (72-120 hours) to allow polysaccharide transfer. Caride and protein consumption was maximized.

[0146] Acid-hydrolyzed serotype 18C was purified to approximately 12.0 g / L and approximately 6.0 g / L of polysaccharide, respectively. Using the alkali and protein concentrations, prepare a solution containing sodium cyanoborohydride and approximately pH 8. Conjugation was performed in 100 mM potassium phosphate at 37°C.

[0147] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L. The solution was cooled to 2-8°C and filtered through a 1.2 micron filter. All serotypes (except serotype 5) were , using a 100 kDa NMWCO tangential flow ultrafiltration membrane, at 2-8 °C in 100 mM phosphate buffer. Diafiltered against potassium chloride, pH 7.0, and then recovered in the retentate. The resulting batch was diluted to approximately 2.0 g polysaccharide / L and diluted with 1.2 M sodium bicarbonate. The pH was adjusted to 9.4 by adding sodium borohydride (polysaccharide 1.5 M potassium phosphate, pH 6.0 was added. Serotype 5 was then added using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltered against 300 mM potassium phosphate.

[0148] Final Filtration and Product Storage The batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 10°C. The retentate batch was filtered to 0.2 microns.

[0149] Serotype 19F was incubated at 22°C for approximately 7 days and subjected to 100 kDa NMWCO tangential flow. Using an ultrafiltration membrane, 10 mM HCl in 150 mM sodium chloride, pH 7.0 at 4 °C. Diafiltered against L-histidine and 0.2 micron filtered.

[0150] Add 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 to the batch. The polysaccharide concentration was adjusted to 1.0 g / L using ethanol. The batch was divided into aliquots. and frozen at ≦−60°C.

[0151] Example 5: CRM using reductive amination in dimethyl sulfoxide 197 Blood to Conjugation of cytochromes 6A, 6B, 7F, 18C, 19A, 19F, and 23F How to Different serotype polysaccharides were purified using a common process flow. 197 The polysaccharides were individually conjugated to carrier proteins. The activated polysaccharide was reduced to its molecular mass, chemically activated, and buffer exchanged by ultrafiltration. Saccharides and purified CRM 197 were individually freeze-dried and dissolved in dimethyl sulfoxide (DMSO). The reconstituted polysaccharide and CRM were then reconstituted in 500 ml of HCl. 197 Solution below The resulting conjugate was combined and conjugated as described above. The solution was purified by ultrafiltration before microfiltration. Some process parameters within each step are constrained to serotype-specific values ​​in the following sections. Controlled.

[0152] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and used for all sera except for serotype 19A. All serotypes except serotypes 18C and 19A were homogenized. The molecular mass of the polysaccharide was reduced by homogenization. and the number of passes through the homogenizer was adjusted to the serotype-specific target (150-1000 bar; 4-7 Serotype 18C was size reduced by acid hydrolysis at ≥90°C. Serotype 19A did not decrease in size.

[0153] The size-reduced polysaccharide was filtered through 0.2 microns and then concentrated to a 10 kDa Diafiltered against water using a NMWCO tangential flow ultrafiltration membrane. MWCO membranes were used for serotype 18C.

[0154] The polysaccharide solution was then diluted with sodium acetate buffer at serotype-specific temperatures ( The temperature was adjusted to 4-22°C and the pH to 4-5. Polysaccharide activation was performed with metaperiodine. The amount of sodium metaperiodate added was determined by the serotype. It is specific and has about 0.1 to 0.5 moles of metathesis per mole of polysaccharide repeating unit. It turned out to be sodium iodate.

[0155] For all serotypes, the activated product was filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltered against 10 mM potassium phosphate, pH 6.4 using 5 kD A NMWCO membrane of 18C was used for serotype 18C. Ultrafiltration for all serotypes was performed at 2–8°C. So I went.

[0156] CRM 197 Polysaccharide conjugation to As previously described (see International Patent Application Publication No. WO 2012 / 173876 A1 Purified CRM obtained through expression in Pseudomonas fluorescens 197 of , against 2 mM phosphate, pH 7 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The mixture was diafiltered and filtered to 0.2 microns.

[0157] The oxidized polysaccharide solution was formulated with water and sucrose in preparation for lyophilization. The protein solution was formulated with water, phosphate buffer, and sucrose in preparation for lyophilization. The lysate concentrations ranged from 1 to 5% and optimal reconstitution was achieved in DMSO after lyophilization.

[0158] Formulated polysaccharides and CRM 197 The solutions were freeze-dried individually. Saccharides and CRM 197 The material was redissolved in DMSO and mixed using a tea mixer. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was mixed. mol) and allow conjugation to proceed for a serotype-specific period (1-48 hours). , achieving the target conjugate size.

[0159] Reduction with sodium borohydride Sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added to the conjugate. The batch was diluted in 150 mM sodium chloride at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was then concentrated. and filtered using a 10 kDa NMWCO tangential flow ultrafiltration membrane in 150 mM sodium chloride. The mixture was diafiltered at approximately 4°C against the lyophilized milk.

[0160] Final Filtration and Product Storage Each batch was then concentrated and filtered using a 300 kDa NMWCO tangential flow ultrafiltration membrane. for 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C The retentate batch was 0.2 micron filtered.

[0161] Serotype 19F was incubated for approximately 5 days and then filtered through a 300 kDa NMWCO tangential flow ultrafiltration filter. Using the membrane, lyse the membrane in 10 mM L-hydroxybenzoates in 150 mM sodium chloride, pH 7.0, at approximately 4 °C. Diafiltered against stigmine and 0.2 micron filtered.

[0162] Add 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 to the batch. The solution was diluted with ethanol, dispensed into aliquots, and frozen at ≤-60°C.

[0163] Example 6: Mouse immunogenicity study using ST3-DTFB monovalent conjugate formulations ST3-CRM 197 The immunogenicity of ST3-DTFB compared with that of The adjuvant formulation for administration to mice was 0.08 μg per 100 μL. For a dose of 100 μg polysaccharide and 5 μg aluminum, 24 μL of sterile filtered Conjugate (1:10 in saline - 0.1258 mg of DTFB or C per mL) RM 197 conjugated polysaccharide) and 62 μL of APA, and 3.664 m The vaccine was prepared by mixing 1 L of sterile saline with 10 mL of PBS. The formulated vaccine was stored at 2-8°C. The cells were stored in individual borosilicate stoppered vials to support individual immunizations.

[0164] ST3-DTFB was evaluated in 6- to 8-week-old female Balb / C mice (n = 10 / Mice were treated with the proprietary ST3-DTFB and DTFB-treated mice, which were prepared as described in Examples 3 and 4. and ST3-CRM 197 ST3-DTFB / A made using conjugate preparation PA and two ST3-CRMs 197 / APA lot. ST3 PnPs The concentrations were 0.1 ml with 5 μg of APA administered intraperitoneally on days 0, 14, and 28. The serum was collected at pre-study and 39-day intervals. The subjects were collected on post-dose 3 (PD3) and analyzed by ST3 WHO ELISA [standardized World Health Organization (WHO) according to WHO protocols] and protein carrier ELISA (CRM 197 oh Mice were tested in the DTFB (and DTFB). E. coli serotype 3 (207 CFU / 0.5 ml) was administered intraperitoneally.

[0165] The WHO ELISA results (Figure 4) showed that ST3-DTFB / APA and ST3-CRM 19 showed that mice immunized with both 7 / APA had similar PD3 PnPs 3 titers. ST3-DTFB / APA and ST3-CRM 197 / APA-immunized mice had ≥90% protection against serotype 3 challenge, which was higher than the negative controls saline and APA The levels were significantly higher than those in immunized mice (Fig. 5).

[0166] Example 7: 15-valent pneumococcal conjugate vaccine with different surfactants and stabilizers Chin's preparation The pneumococcal polysaccharide-protein conjugates prepared as described above were administered to the blood. Clear type 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F 15-valent pneumococcal conjugate vaccine (PCV15) with , 23F, and 33F The formulations were prepared by reducing the hydroxybenzoates in aqueous solution (Example 4) or in DMSO (Example 5). Pneumococcal polysaccharide-CRM produced by selective amination 197 Conjugates The ST3-DTFB conjugate was prepared according to Example 3. The required dose of bulk conjugate required to achieve the target final concentration of each serotype was calculated. Calculated based on the suction volume and bulk polysaccharide concentration. The body was treated with PS-20, PS-80, or P188 in combination with sodium chloride, L-histidine, , and a pH 5.8 buffer solution.

[0167] Sterile formulation bulk is prepared using propylene glycol (PG) and polyethylene glycol 4 00(PEG 400 ) with or without bulk aluminum phosphate adjuvant ( The two concentrations of conjugates were mixed gently during and after blending with the APA. The conjugates and APA were studied in various formulations. One was serotype 1 at 8 μg / mL. Contains 6B polysaccharide and 4 μg / mL polysaccharide for all other serotypes One contained 16 μg / mL serum and 250 μg / mL APA. type 6B polysaccharide, 8 μg / mL polysaccharide for all other serotypes; The formulated vaccine was stored at 2-8°C. Ta.

[0168] Example 8: Conjugates containing conjugates produced by reductive amination in aqueous solution Effect of excipients on the stability of pneumococcal conjugate vaccine formulations 15-valent pneumococcal conjugate vaccine (PCV1) prepared as described in Example 7 5) The stability of was evaluated for various excipient conditions after agitation, recirculation, and rotary agitation studies. PCV15 was incubated at 20 mM L -histidine, pH 5.8, 150 mM sodium chloride, and the Two concentrations of either the conjugate or APA were used. The results were very similar between the two formulations at different concentrations of conjugate and APA. , Results for PCV15 formulations containing lower concentrations of conjugate and APA For the agitation study, the PCV15 formulation was electrochemically mixed in a glass container. For recirculation and shear testing, the PCV15 formulation was placed in a tube. For the rotation test, the P was recirculated in a group as outlined in Example 7 and Table 2. Prepare the CV15 base formulation (L-histidine, pH 5.8, and sodium chloride). Surfactants or stabilizers were added. Agitation studies were performed with rotary side agitation at 4°C for up to 24 hours. The formulation was evaluated using visual evaluation. The path of the light beam through the container was Furthermore, the effects of manufacturing, transportation, and handling on particle size distribution were investigated. The effect of loading stress was assessed using static light scattering (SLS). Static light scattering of the product allows for more sensitive detection of agglomeration as indicated by particle size distribution. The monodisperse (unimodal) particle size distribution of the PCV15 drug product indicates a non-aggregated drug product. However, the polydisperse multimodal particle size distribution indicates aggregation.

[0169] Stability of PCV15 in agitation studies CRM was prepared using reductive amination under aqueous conditions as described in Example 4. 197 ( PCV15 Aq Streptococcus pneumoniae polysaccharide biologics conjugated to The PCV15 formulation was mixed in the laboratory-scale mixing equipment (beaker and magnetic stir bar) described above. PCV15 was screened using Aq used reductive amination under aqueous conditions. Use CRM 197 Conjugated Streptococcus pneumoniae serotype 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 Contains polysaccharides from F, and 33F. 20 mM L-hybrid with APA PCV15 in 150 mM NaCl, using a beaker and a magnetic stir bar In the absence of surfactant, PCV15 Aq The formulation is susceptible to manufacturing (agitation) induced damage, which may affect the quality of the vaccine drug product. During agitation to ensure uniformity, clumping of the vaccine drug product may occur.

[0170] A class of non-ionic triblock copolymers is currently being studied for screening various excipients. Poloxamer 188 (P188) was selected and found to provide robust stability to the This resulted in controlled aggregation and provided a robust and stable vaccine drug product formulation. Aq Made The reagent was prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl, APA, and No P188, 0.08 w / v% P188, or 0.24 w / v% P188 The effect of time under constant stirring using a magnetic stir bar was investigated using static light scattering. The particle size distribution was evaluated using a Malvern Mastesizer (Figures 6-7). 2000. The expected particle size distribution was measured using a 5 μm NIST particle size standard. For all formulations (with or without poloxamer 188), AP A monodisperse histogram profile was observed after addition of the conjugate to A (T = 0 hours of mixing). Formulations without P188 after only 7 hours of continuous mixing (T = 7 hours of mixing). The results showed an increased particle size distribution and the appearance of agglomerates (Figure 6). Formulations containing P188 at concentrations of 0.01% or less showed increased particle size upon continuous mixing for up to 24 hours. The results showed no evidence of aggregation or the appearance of aggregation (T = 24 hours of stirring) (Figure 7).

[0171] Stability of PCV15 in horizontal agitation studies in 20 mM L-histidine, pH 5.8, 150 mM NaCl, and APA, Additional PCV15 with or without 0.2 w / v% P188 Aq The formulations are shown in Example The samples were prepared in 100 mL laboratory-scale batches as described in 7. To simulate handling and evaluate the impact on vaccine drug product stability, The study utilized a stirring test. The test was conducted on the surface of a closed container system (syringe or vial). PC through interactions with and exposure of the formulation to final container components and air interfaces V15 Aq This represents direct mixing of the formulation. 0.64 mL was dispensed into syringes and capped. The syringes were rotated horizontally at 2-8°C for 24 hours and evaluated for particle size distribution using SLS. (Figure 8A). Visual evaluation was also performed (Figure 8B). PCV15 in the absence of provided P188 Aq The formulation is characterized by an increase in particle size distribution of the drug product and This can result in visible signs of agglomeration and clumping in container closures such as syringes. The PCV15 formulations did not show any visual signs of increased particle size distribution or agglomeration and aggregation. Ta.

[0172] Example 9: Conjugates produced by reductive amination in aqueous and DMSO solutions Safety of pneumococcal conjugate vaccine drug products prepared using a mixture of conjugates Effect of excipients on stabilization Complex with APA in 20 mM L-histidine, pH 5.8, 150 mM NaCl Several 15-valent (PCV15) formulations were evaluated using a laboratory-scale simulated transport study to demonstrate robust A manufacturable and commercially viable vaccine drug product formulation was ensured. Aq teeth Streptococcus pneumoniae polysaccharide-CRM1 produced by reductive amination in aqueous solution 97 The formulation contained the conjugate PCV15. Aq / Non-Aq in DMSO Serotypes 6A, 6B, 7F, 19A, 19F, and 19C were prepared using reductive amination of and 23F conjugates, as well as those prepared using reductive amination in aqueous solution. Serotypes 1, 3, 4, 5, 9V, 14, 18C, 22F, and 33F conjugates wherein all polysaccharides are CRM 197 was conjugated to PCV15 formulation Aq / Non-Aq / sT3-oTFB serotype 3 is CRM 197 in PCV15 except that it is conjugated to the DTFB protein rather than the IgG. Aq / Non-Aq The results were similar to those of the conjugates at different concentrations listed in Example 7. The results for the two formulations in the body and APA were very similar, so unless otherwise stated, Only results for PCV15 formulations containing low concentrations of conjugate and APA are presented. Prevnar 13® is used as another example of a polyvalent formulation as shown in this example. to test the compatibility of PS-80 in different pneumococcal conjugate vaccine products. That is CRM 197 Streptococcus conjugated to a carrier protein Pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A , 19F, 23F polysaccharides, polysorbate 80, succinate buffer and phosphate The conjugate contains an aluminum acid adjuvant. or reductive amination under aqueous conditions.

[0173] A horizontal agitation study was used to assess the impact on the stability of the vaccine formulation. is the interaction with the surface in the container closure (syringe or vial) and the final This represents direct agitation of the formulation through exposure of the formulation to the container components and air interface. These syringes were stored at 2-8°C for up to 8 hours. The effect of time under horizontal agitation was investigated by measuring particle size using static light scattering (SLS). The particle size and distribution were evaluated using a Malvern Mastesizer 20 The particle size distribution was evaluated using a 5 μm NIST particle size standard. As shown in Figure 9, P188 was Aq Robust stabilizer for formulations Nevertheless, PCV15 Aq / Non-Aq / ST3-DTFB Regarding formulations It was not an effective stabilizer for controlling aggregation. It increased the particle size distribution as well as agglomerates and Visible signs of agglutination and aggregation were observed in PCV15 with P188. Aq / Non-Aq / sT3-oT FB It was noted in the formulation.

[0174] P188 contains the conjugate produced by reductive amination in DMSO. The surprising finding that the FDA did not provide robust stability to PCV15 formulations with additional PCV15 stabilizers and excipients were screened. Aq / Non-Aq / ST3- DTFB Formulation (AP in 20 mM L-histidine, pH 5.8, 150 mM NaCl) A and various stabilizers) into syringes and screened using horizontal agitation. The effect of time under constant horizontal rotation was assessed using visual assessment (Table 2). [Table 3]

[0175] PCV15 Aq / Non-Aq / ST3-DTFB Visual evaluation of the formulations showed a higher P18 8 and PS-20 concentrations showed no signs of aggregation (Table 2). (0.05 w / v% to 1.0 w / v%) or PS-20 (0.005 w / v% to 0.1 PCV15 containing 100% (w / v%) Aq / Non-Aq / ST3-DTFB Formulation invisible to the naked eye Higher resolution studies to evaluate the optical particle size distribution were carried out using SLS. The formulation was dispensed into a 1.5 mL HyPak syringe (Becton-Dickinson). The D[4,3] values ​​measured by SLS are shown in Figure 10A-B. The results of D[4,3] show that higher concentrations of PS-20 significantly improved the physicochemical stability of the formulation. Figure 1 shows that although there was a slight improvement, P188 at all concentrations was not effective. 10B, horizontal agitation of Prevnar 13® for up to 24 hours. Agitation showed an increase in D[4,3] values, evidence of particulates, demonstrating that the formulation was resistant to simulated transport-induced aggregation. was not sufficiently protected, similar to our own experience using PS-80 in our formulations. Showing that they were similar.

[0176] Based on our data using PS-20 and PS-80, Other pneumococcal polysaccharides containing one or more conjugates made with Improved stability is expected with do-protein conjugates.

[0177] Example 10: Use of ST3-DTFB conjugate formulated in PCV15 Infant Rhesus Macaque (IRM) immunogenicity study DTFB (multimodal cation exchange chromatograph) prepared as described in Example 1 using fluoromography to obtain the ST3-DTFB conjugate as described in Example 3. The PCV15 formulation was prepared as in Example 7. Each group (n=8 / group) received 100 μL of ... The animals were immunized intramuscularly (arms 1-4) with the L vaccine. Serum was collected before the study started (pre-study) and at 14 and 2 IRM were collected on days 8, 42, 56, and 70. IRM were monitored for any signs of illness or distress. The animals were observed twice daily by trained animal care staff. was deemed safe and well tolerated, with no vaccine-related adverse events observed. Ta. [Table 4]

[0178] The mouse studies described in Example 6 were performed with a monovalent conjugate containing a single PnPs serotype 3. A WHO ELISA was completed to assess IgG responses. To assess serotype-specific IgG responses in the 2-valent vaccine, multiplexed electrochemiluminescence (EC) L) Assays were performed using SULFO-TAG™ labels that emit light upon electrochemical stimulation. The MSD technology used (MSD is MesoSca, Gaithersburg, Maryland, USA) MesoScale Disc, a division of le Diagnostics, LLC. (a trademark of Overy) was used to Based on the SEQ ID NO: 1, human antibody reagents and standards were developed for use with rhesus monkey serum. was used when testing pup samples. The results from pup rhesus monkeys were compared with the human reference standard (007 The serotype-specific IgG concentrations assigned to each serotype (sp) were used to calculate the number of serotypes read from the standard curve. Expressed as mean concentration.

[0179] Serotype 3 post-dose 3 (PD3) IgG responses (Figure 11) showed no statistically significant differences between immunization groups. No difference was observed between the OPA assay and the Streptococcus pneumoniae serotype 3 assay. Functional antibodies assessed in the immunization group (Fig. 12) did not show any statistical differences between the immunization groups. There wasn't.

[0180] Example 11: Optimization of PCV15 formulation The results are shown for two preparations at different concentrations of conjugate and APA as listed in Example 7. The results were very similar between the conjugates, so unless otherwise stated, the lower concentrations of the conjugates were used. Only results for PCV15 formulations containing PEG and APA are presented in this example.

[0181] A recirculation line is used in the PCV15 formulation process to convert the vaccine drug product into syringes. The recirculation during filling is common in biotherapeutic drug products. These shear forces and stresses are then applied, and therefore important processing steps to evaluate. A study was conducted to compare the effects of 0.2 w / v% P188 and 0.1 w / v% PS- PCV15 containing 20 Aq / Non-Aq / ST3-DTFB and formulation (64 μg 20 mM L-hydroxybenzoates with 250 μg / mL total polysaccharides and 250 μg / mL APA The effect of recirculation on the production of estradiol (stigmine, pH 5.8, 150 mM NaCl) was evaluated. The agent was pumped from the supply container through the tubing at a flow rate of 180 mL / min using a peristaltic pump for 24 hours. The feed vessel was continuously mixed using a magnetic stir bar and the sample was taken for visual observation. The formulations containing P188 showed the appearance of clumps or visual aggregates. (Table 4), but the formulation containing PS-20 showed no visible signs of aggregation. [Table 5]

[0182] Additional recirculation tests were performed up to 500 μg / mL (w / v Al +3 ) APA and P1 PCV15 with 88 or PS-20 Aq / Non-Aq and PCV15 Aq / N on-Aq / ST3-DTFB The formulation was tested for 24 hours with constant mixing. After recirculation, the formulation was dispensed into syringes and stirred horizontally for up to 24 hours. A summary of the visual evaluation of the gel is shown in Table 5. These results indicate that PS-20 is suitable for conventional manufacturing and and robust protection against physical instability or agglomeration that may occur during transportation and handling. P188 is a PCV15 Aq in formulation stabilization Despite the success (Figures 7-9), this PCV15 Aq / Non-Aq / ST3-DTF B The formulation was not able to provide a satisfactory stability profile. [Table 6]

[0183] Further studies were carried out to prepare ST18C using reductive amination in DMSO. PCV15 manufactured Aq / Non-Aq / ST3-DTFB Regarding the formulation (PCV1 5 Aq / Non-Aq / ST3-DTFB / ST18C-Non-Aq ). PCV15 A q / Non-Aq / ST3-DTFB / ST18C-Non-Aq The formulation was administered at 64 μg / m L total polysaccharides, 250 μg / mL APA, and 0.2 w / v% PS-2 Prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl with 0 PCV15 Aq / Non-Aq / ST3-DTFB / ST18C-Non-Aq The formulation After recirculation with constant mixing for up to 6 hours, the formulation is dispensed into syringes and stored for up to 24 hours. The formulations were inspected and a summary of the visual evaluation of the syringes is shown in Table 6. The results showed that PS-20 effectively inhibited the growth of 250 μg / mL APA and 0.2 w / v% PS-20. Prepared in 20 mM L-histidine, pH 5.8, 150 mM NaCl with D Pneumococcal polysaccharide serotype 6A prepared using reductive amination in MSO , 6B, 7F, 18C, 19A, 19F, and 23F conjugates in aqueous solution. Serotypes 1, 3, 4, 5, 9V, 14, 22F, and 16F were prepared using reductive amination of and 33F conjugates, wherein all polysaccharides are CRM 197 For preparations containing PCV15 conjugated to and a robust solution to physical instability or clumping that may occur during handling. It indicates that measures are being offered. [Table 7]

[0184] As shown in Figure 10 and Tables 4-5, P188 alone inhibited the reduction of amino acids in DMSO. Conjugates and / or ST3-DTFB conjugates produced using nitration Minimal benefit in preventing aggregation of suspension-based PCV15 formulations consisting of hydroxybenzoates. Additional mixing and horizontal rotation studies were performed with 0.2% w / v P188 and PE G 400 with formulations containing PEG or propylene glycol (PG) stabilizers I went. PCV15 Aq / Non-Aq / ST3-DTFB The formulation was as described in Example 7. PEG was prepared on a 500 mL scale. 400 Alternatively, PG is added to APA, and then The conjugate was added: 64 μg PnPs / mL, 250 μg / mL APA, and PEG in a formulation containing 0.2 w / v% P188 400 or final concentration of PG The formulation was stirred continuously for 1 hour using a magnetic stirrer. The mixture was mixed and dispensed into syringes. The syringes were agitated horizontally for up to 24 hours. The formulations were visually evaluated and Syringes were sampled periodically for particle size distribution measurement by SLS and visual evaluation of the syringes. The results are shown in Tables 7 and 8. The particle size distribution results are shown in Figure 13. When added alone, P18 8 did not control aggregation in the formulation. 400 or PG, P When combined with the 188 it provided sufficient stability. [Table 8] [Table 9]

[0185] Example 12: Immunogenicity of stabilized formulations of pneumococcal conjugate vaccines Optimization to control manufacturing and transport-induced instability on immunogenicity in pups of rhesus monkeys The effects of the PCV15-containing formulation prepared in Example 11 were evaluated. As described above, 64 μg PnPs / mL, 20 mM L-histidine, pH 5.8, 1 50 mM sodium chloride, 250 μg / mL APA, and 0.2 w / v% P18 8, 0.0 containing either 15 w / v% PG or 0.1 w / v% PS-20. 1 mL of PCV15 Aq / Non-Aq / ST3-DTFB The formulation was injected intramuscularly. were performed at ages T=0 (Dose 1), 1 month (Dose 2), and 2 months (Dose 3). Serum The results were collected before dose 1 and 2 weeks after doses 1, 2, and 3. Serum IgG levels from post-dose 2 and post-dose 3 serum samples were measured as described in Example 10. The results shown in Figure 14 showed that the effect of either PS-20 or the combination of P188 and PG was This indicates that the PCV15 formulation with

[0186] Example 13: Reductive HCl in protic (water) and aprotic (DMSO) solutions Pneumoniae prepared using different mixtures of conjugates produced by amination Effect of polysorbate 20 and polyisobutene on the stabilization of coccal conjugate vaccine drug products. The influence of Rubet 80 The encouraging results seen with the PCV15 formulations in the above examples were due to the fact that they were made in DMSO. Protein levels in glycoconjugates prepared in aqueous conditions versus those prepared in aqueous conditions The exploration of the lower and upper limits of the ratio of glycoconjugates is warranted. The formulations consisted of pneumococcal polysaccharides produced by reductive amination in aqueous solution or DMSO. Saccharide-CRM 197 Each formulation contained a conjugate of 20 mM L-histidine, pH 5.8, 150 mM NaCl, normalized to 64 μg / mL (w / v Ps) total polysaccharides with 50 μg / mL APA (Ps) concentration. PS-80 or PS-20 was added to each formulation. (0.05w / v%) or PS-20(0.05w / v%) or PS-20(0.2 The formulations were delivered in BD HyPAK prefilled syringes to achieve a final concentration of either 0.01% w / v or 0.1% w / v. The mixture was dispensed into syringes and evaluated using laboratory-scale simulated transport studies to identify a robust, manufacturable, and commercially available This ensured a viable vaccine drug product formulation.

[0187] To achieve the desired range of percentages of glycoconjugates prepared using DMSO In the present study, all polysaccharides were synthesized using reductive amination. 197 Conjugated to The following formulations were prepared:

[0188] Preparation PCV 24% serotypes 6A, 6B, and 23F conjugates prepared in DMSO Conjugates and serotypes 1, 3, 4, 5, 7F, 9V, 14, and 1 prepared in aqueous solution This formulation contained 8C, 19A, 19F, 22F, and 33F conjugates. The total protein in the sera was 56 μg / mL, which is 13 μg / mL, or approximately 24% of the total protein. Proteins were conjugated to polysaccharides using reductive amination in DMSO. CRM 197 It was made up of:

[0189] Preparation PCV 50% serotypes 6A, 6B, 7F, 19A, and 19C prepared in DMSO. F, and 23F conjugates, and serotypes 1, 3, 4, and 5 prepared in aqueous solution This formulation contained 9V, 14, 18C, 22F, and 33F conjugates. The total protein in the sera was 62 μg / mL, which is 31 μg / mL, or 50% of the total protein. The protein was conjugated to a polysaccharide using reductive amination in DMSO. CRM 197 It was made up of:

[0190] Preparation PCV 62% serotypes 6A, 6B, 7F, 19A, and 19C prepared in DMSO. F, and 23F conjugates, and serotypes 1, 5, and 9V prepared in aqueous solution. The total amount of 14, 18C, 22F, and 33F conjugates in this formulation was Protein was 61 μg / mL, and 38 μg / mL, or approximately 62% of the total protein was D CRM conjugated to polysaccharides using reductive amination in MSO 197 It was made up of:

[0191] Preparation PCV 79% serotypes 6A, 6B, 7F, 19A, and 19C prepared in DMSO. F, and 23F conjugates, and serotypes 1, 5, and 18C prepared in aqueous solution The total protein in this formulation was 63 μg / mL, and 50 μg / mL, or approximately 79% of the total protein, was reduced in DMSO. CRM conjugated to polysaccharides using methylation 197 It was made up of:

[0192] Preparation PCV 100%Serotype 6A prepared using reductive amination in DMSO , 6B, 7F, 19A, 19F, and 23F conjugates. The total protein in the 65 μg / mL sample was 65 μg / mL, or 100% of the total protein. CRM in which proteins were conjugated to polysaccharides in DMSO 197 It was made up of:

[0193] Horizontal stirring studies were used to characterize the synthesis of tannins conjugated to polysaccharides in DMSO. The effect of protein to total protein ratio on product stability was evaluated. Interaction with surfaces in the chain system (syringe or vial) and final container configuration This represents direct agitation of the formulation through exposure of the formulation to the air and air interface. These syringes were stored horizontally at 2-8°C for up to 24 hours. The effect of time under horizontal agitation was investigated on particle size distribution using static light scattering (SLS). Particle size and distribution were evaluated using a Malvern Mastesizer 2000. A 5 μm NIST particle size standard was run to generate the expected particle size distribution. As shown in Figures 15A-E, PS-80 was effective against all PCV-containing drug products (PCV 24 % ~PCV 100% ) was not an effective stabilizer for controlling aggregation. Increased distribution and visible signs of agglomeration (appearance of particles) and aggregation with PS-80 This was observed for all formulations.

[0194] Surprisingly, PS-20 at concentrations similar to those used in PS-80 did not produce any of the compounds tested. Improved stability profile across a range of DMSO conjugate percentages The PS-20 solution was added to achieve a concentration of 0.2% PS-20 in the formulation buffer. Addition of 0 resulted in a significant increase in the DMSO conjugate percentage across the range of DMSO conjugate percentages tested. Superior stability was obtained compared to 0.05% PS-20.

[0195] Example 14: Safety of pneumococcal conjugate vaccine in New Zealand White rabbits Immunogenicity of fixed formulations Controlling manufacturing and transport-induced instability on immunogenicity in New Zealand White rabbits The effects of a PCV15-containing formulation optimized for the treatment of rhesus mastitis were evaluated. As described in Example 11, 64 mg PnPs / mL, 20 mM L-histidine, p H5.8, 150 mM sodium chloride, 250 μg / mL APA, and 0.2 w / 0.1 mL of PCV15 containing v% PS-20 Aq / Non-Aq Intramuscular injection of the preparation Injections were given on days 0 and 14. Sera were collected from the vaccinated animals on days 0 and 14. Serum was collected pre-immunization, post-dose 1, and post-dose 2 serum samples. IgG levels were determined by ECL analysis.

[0196] The results shown in Figure 16 show that the saccharides were 4 μg / mL, except for 6B at 8 μg / mL, and the CRM was approximately 64 μg / mL. 197 CRM using reductive amination in DMSO, formulated into a dosage form containing a carrier protein 197 Streptococcus pneumoniae polysaccharides from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugated to CRM using reductive amination in aqueous solution 197We demonstrate that a 15-valent pneumococcal conjugate formulation in 20 mM histidine pH 5.8, 150 mM NaCl, 250 μg / mL APA, 0.2 w / v% PS-20, with Streptococcus pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F conjugated to Streptococcus pneumoniae is immunogenic. In one aspect, the present invention provides the following. [Item 1] 1. A formulation comprising: (i) one or more polysaccharide-protein conjugates; (ii) a pH-buffered saline solution having a pH in the range of 5.0 to 7.5; (ii) an aluminum salt; and (iv) a surfactant system selected from a) polysorbate 20 and (b) a poloxamer having a molecular weight in the range of 1100 Da to 17,400 Da and a polyol selected from propylene glycol and polyethylene glycol 400. [Item 2] 10. The formulation of claim 1, wherein one or more of the polysaccharide-protein conjugates are made in an aprotic solvent. [Item 3] 2. The formulation of item 1, wherein the polysaccharide-protein conjugate is prepared in an aprotic solvent at 10-100% by weight protein. [Item 4] 4. The formulation of item 2 or 3, wherein the aprotic solvent is DMSO. [Item 5] 2. The formulation of item 1, wherein the surfactant system comprises a poloxamer having a molecular weight in the range of 1100 Da to 17,400 Da. [Item 6] 6. The formulation of item 5, wherein the poloxamer has a molecular weight in the range of 7,500 Da to 15,000 Da. [Item 7] 6. The formulation of item 5, wherein the poloxamer has a molecular weight in the range of 7,500 Da to 10,000 Da. [Item 8] 5. The formulation according to any one of items 1 to 4, wherein the poloxamer is poloxamer 188 or poloxamer 407. [Item 9] 9. The formulation according to any one of items 1 to 8, wherein the final concentration of the poloxamer is 0.001% w / v to 5% w / v. [Item 10] 10. The formulation of item 9, wherein the final concentration of the poloxamer is 0.025% w / v to 1% w / v. [Item 11] 11. The formulation according to any one of items 1 to 10, wherein the polyol is propylene glycol at a final concentration of 1% to 20% w / v. [Item 12] 11. The formulation according to any one of items 1 to 10, wherein the polyol is polyethylene glycol 400 at a final concentration of 1% to 20% w / v. [Item 13] 2. The formulation of item 1, wherein the surfactant system comprises polysorbate 20. [Item 14] Item 14. The formulation according to item 13, wherein the final concentration of polysorbate 20 is in the range of 0.001% w / v to 10% w / v. [Item 15] Item 14. The formulation according to item 13, wherein the final concentration of polysorbate 20 is in the range of 0.025% w / v to 2.5% w / v. [Item 16] Item 14. The formulation according to item 13, wherein the final concentration of polysorbate 20 is in the range of 0.025% w / v to 0.1% w / v. [Item 17] 17. The formulation according to any one of items 13 to 16, further comprising a polyol selected from propylene glycol and polyethylene glycol. [Item 18] Item 18. The formulation according to item 17, wherein the polyethylene glycol or propylene glycol is at a final concentration of 6% w / v to 20% w / v. [Item 19] 19. The formulation of item 18, wherein the polyethylene glycol is polyethylene glycol 400. [Item 20] 20. The formulation according to any one of items 1 to 19, wherein the pH-buffered saline has a pH in the range of 5.0 to 7.0. [Item 21] 21. The formulation of item 20, wherein the buffering agent is selected from the group consisting of phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate, and citrate. [Item 22] 22. The formulation of item 21, wherein the buffering agent is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM. [Item 23] 23. The formulation of item 22, wherein the L-histidine is at a final concentration of 20 mM ± 2 mM. [Item 24] 24. The formulation of any one of items 1 to 23, wherein the salt in the pH-buffered saline is magnesium chloride, potassium chloride, sodium chloride, or a combination thereof. [Item 25] 25. The formulation of item 24, wherein the salt in the pH buffered saline is sodium chloride. [Item 26] 26. The formulation according to any one of items 1 to 25, wherein the saline solution is present in a concentration of 20 mM to 170 mM. [Item 27] 27. The formulation of any one of items 1 to 26, wherein the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides conjugated to a carrier protein. [Item 28] The carrier protein is a CRM 197 28. The formulation according to item 27, wherein the exotoxin is selected from diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, Escherichia coli LT, Escherichia coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof. [Item 29] One or more of the polysaccharide-protein conjugates is a CRM 197 29. The formulation of item 28, wherein the [Item 30] The polysaccharide-protein conjugate formulation is a CRM 197 30. The formulation of item 29, which is a 15-valent pneumococcal conjugate (15vPnC) formulation consisting essentially of Streptococcus pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to [Item 31] 31. The formulation of item 30, wherein one or more of the polysaccharide-protein conjugates are prepared using reductive amination under DMSO conditions. [Item 32] 32. The formulation of item 31, wherein polysaccharide-protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared under DMSO conditions, and polysaccharide-protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous conditions. [Item 33] Each dose contained 4 μg / mL or 8 μg / mL of each saccharide, except for 6B, which was 8 μg / mL or 16 μg / mL, and approximately 64 μg / mL or 128 μg / mL of CRM. 197 33. The formulation of item 32, formulated to contain a carrier protein. [Item 34] 34. The formulation of item 33, further comprising 20 mM L-histidine, pH 5.8, 150 mM sodium chloride, 0.25 mg / mL aluminum phosphate adjuvant (APA), and 0.2 w / v% PS-20. [Item 35] CRM 197 1. A formulation comprising a 15-valent pneumococcal conjugate composition consisting essentially of Streptococcus pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to 20 mM histidine, pH 5.8, 150 mM NaCl, 250 μg / mL APA, and 0.2 w / v% PS-20, wherein the formulation contains 4 μg / mL of each saccharide except for 6B at 8 μg / mL, and about 64 μg / mL CRM. 197 The polysaccharide-protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared under DMSO conditions, and the polysaccharide-protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous conditions.

Claims

1. (i) one or more polysaccharide-protein conjugates; (ii) a pH buffered saline solution having a pH in the range of 5.0 to 7.5; (iii) an aluminum salt; and (iv) Polysorbate 20 (PS-20) A formulation comprising: the polysaccharide is a S. pneumoniae polysaccharide; the protein is CRM197, 100% of the conjugate (based on total protein) is prepared in dimethyl sulfoxide (DMSO); The preparation, wherein the concentration of PS-20 is 0.2 w / v%.

2. 2. The formulation of claim 1, wherein the aluminum salt is aluminum phosphate adjuvant (APA).

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