Nanoemulsion adjuvant composition for pneumococcal conjugate vaccine

Abstract

The present invention provides a composition to be administered as a vaccine for the prevention of pneumococcal disease. [Solution] A pneumococcal conjugate composition comprising a Streptococcus pneumoniae polysaccharide-carrier protein conjugate and a stable nanoemulsion (SNE) is provided, wherein the SNE comprises sorbitan trioleate, polysorbate-20 or polysorbate-80, and squalene.

Description

[Technical Field]

[0001] This invention is a nanoemulsion for pneumococcal conjugate vaccines. This relates to adjuvant compositions. [Background technology]

[0002] Pneumococcal disease is caused by the bacterium Streptococcus pneumoniae. Pneumococcus is an infectious disease caused by the bacterium *Streptococcus pneumoniae*. It is known that different bacterial serotypes can cause different disease symptoms, and infections can occur in the ear. It can cause a variety of symptoms, from sinus infections to pneumonia and bloodstream infections. (Pneumococcal) The disease exhibits high associated morbidity and mortality rates worldwide, particularly among the elderly and children. Currently, 100 capsule polysaccharides have been identified (Ganaie, F. et al. (2020) Clin ical Science and Epidemiology,Vol.11,Iss (ue 3, pp. 1-15). These serotypes are distinguished by their chemical structure, serological response, and other factors. They are distinguished by related genetic mutations.

[0003] In 1983, PNEUMOVAX (registered trademark), a 23-valent pneumococcal vaccine, was introduced. rck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA) has acquired the rights in the United States. It was approved. This vaccine showed reduced immunogenicity in infants through a T-cell-independent response. To address this problem, especially in infants, polysaccharides were covalently bonded to carrier proteins. As a result, the immunogenic response of the pneumococcal conjugate vaccine became T cell-dependent. PCV was developed. In 2000, PR, a 7-valent pneumococcal conjugate vaccine, was developed. EVNAR (Registered Trademark) (Wyeth Pharmaceuticals LLC, Co.) Illegeville (PA) was approved in the United States. In 2010, the 13-valent pneumococcal conjugate was approved. PREVNAR13 (registered trademark), a jugate vaccine (Wyeth Pharma) Ceuticals LLC (Collegeville, PA) has been approved in the United States. In 2021, VAXNEUVANCE, a 15-valent pneumococcal conjugate vaccine, was released. (Registered Trademark) (Merck Sharp & Dohme Corp., a subsidiary) iary of Merck & Co.,Inc.,Kenilworth,NJ,U SA), and PREVNAR20 (registered), a 20-valent pneumococcal conjugate vaccine. Trademark)(Wyeth Pharmaceuticals LLC,Collegevil le,PA) was also approved in the United States. Other polyvalent PCVs are also known and approved worldwide. ru.

[0004] Approved PCV is currently used as an adjuvant to enhance immunogenicity. It utilizes aluminum-containing derivatives. The aluminum adjuvant inhibits the immunogenic response from baseline. Even if it is enhanced, the immunogenic response, especially in infants, is at higher valencies of PCV. It is unclear whether it is sufficient. Therefore, the current aluminum adjuvant standard... Compared to this, identify other adjuvants that can enhance immunogenicity in the case of polyvalent PCV. It is needed. [Overview of the initiative]

[0005] Summary of the Invention This invention relates, in general, to the prevention of pneumococcal disease. More specifically, this invention relates to pneumonia A vaccine containing a coccus conjugate and a stable nanoemulsion (SNE) adjuvant formulation. This disclosure relates to compositions administered as tin. This disclosure, in particular, relates to emulsifiers and / or solubilators. Pneumococcus conjugate containing a surfactant and / or surfactant and / or lipid The present disclosure provides gate compositions, in particular surfactants and / or terpenes and Pneumococcus conjugate containing SNEs containing / or cationic lipids or mixtures thereof This disclosure provides a sorbitan ester and / or terpene composition. This disclosure provides, in particular, a sorbitan ester and / or terpene composition. SNE containing septembryonic acid and / or cationic lipids or mixtures thereof, including Streptococcus pneumoniae This disclosure provides a jugate composition. This disclosure, in particular, provides sorbitan esters, especially polysorbates. Beth-20 or polysorbate-80 or poloxamer, and / or terpe SNE containing septembryonic acid and / or cationic lipids or mixtures thereof, including Streptococcus pneumoniae This disclosure provides a jugate composition. This disclosure, in particular, provides sorbitan esters, especially polysorbates. Beth-20 or polysorbate-80 or poloxamer, and / or terpe SNE containing septembryonic acid and / or cationic lipids or mixtures thereof, including Streptococcus pneumoniae This disclosure provides a jugate composition. This disclosure, in particular, provides a sorbitan trioleate (SPA N-85), polysorbate-20 or polysorbate-80, terpenes and, if desired Pneumococcal conjugate composition containing cationic lipids that may be used more This disclosure provides, in particular, sorbitan trioleate (SPAN-85), poly Sorbate-20 or polysorbate-80, squalene, and optionally used Provided is a pneumococcal conjugate composition comprising an SNE comprising an optional cationic lipid. The present disclosure provides, inter alia, a pneumococcal conjugate composition comprising an SNE comprising sorbitan trioleate (SPAN-85), polysorbate-20 or polysorbate-80, squalene, and a cationic lipid. The present disclosure further provides, inter alia, a pneumococcal conjugate composition comprising an SNE adjuvant formulation comprising 1) sorbitan tri oleate (SPAN-85), 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80), 3) squalene, and 4) an optional cationic lipid. Specific pneumococcal conjugate compositions comprise an SNE adjuvant formulation comprising 1) sorbitan trioleate (S PAN-85), 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80), 3) squalene, and 4) the cationic lipid (13Z,16Z)-N, N-dimethyl-3-nonyldocosa-13,16-dien-1-amine ("CLA"; or "CLA-SNE" when the cationic lipid is comprised in the SNE). Specific pneumococcal conjugate compositions comprise an SNE adjuvant formulation comprising 1) sorbitan triole ate (SPAN-85), 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80), 3) squalene, and 4) the cationic lipid (13Z,16 Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine ("CLA"; or "CLA-SNE" when the cationic lipid is comprised in the SNE). Specific pneumococcal conjugate compositions comprise an SNE adjuvant formulation comprising 1) sorbita n trioleate (SPAN-85), 2) polysorbate-20 (PS-20) or polysorbate-80 (PS-80), 3) squalene, and 4) the cationic lipid (13Z,16 Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine ("CL A"; or "CLA-SNE" when the cationic lipid is comprised in the SNE). Specific pneumococcal conjugate compositions comprise an SNE adjuvant formulation comprising 1) sorbita 1) Polysorbate-20 (PS-20) or 3) Polysorbate-80 (PS-80), 4) Squalene, and 5) Cationic lipids (1 3Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine ("CLA"; or "CLA-SNE" if cationic lipids are included in SNE) Includes an SNE adjuvant formulation containing ''). Specific pneumococcal conjugate compositions include 1 ) Sorbitan Triolet (SPAN-85), 2) Polysorbate-20 (PS-2 0), 3) squalene, and 4) cationic lipid (13Z,16Z)-N,N-dimethyl Lu-3-nonyldocosa-13,16-diene-1-amine ("CLA"; or in SNE) If cationic lipids are present, then SNE adjuvants containing "CLA-SNE" are used. Contains the following: 1) Adjuvant-free pneumococcal conjugate composition, 2) Aluminum phosphate 3) Pneumococcal conjugate composition containing umm adjuvant (APA), or LNP adjuvant. Compared to the performance of the pneumococcal conjugate composition containing Streptococcus aureus, the described SNE The Juvant pneumococcal conjugate composition is effective for most of the tested pneumococcal serotypes. This resulted in an equivalent or enhanced immunogenic response. [Brief explanation of the drawing]

[0006] [Figure 1] Selected structures of cationic lipids: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA); (6Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29-tetraen-18-amine (CLX); and N,N-dimethyl-1-((1S,2R)-2-octylcyclopropyl)heptadecane-8-amine (CLY). [Figure 2] CLA-SNE components: (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA), SPAN-85, PS-20, and squalene. [Figure 3] Characterization of CLA-SNE adjuvant bulk formulations using static light scattering (SLS). See Example 3. [Figure 4] The effect of the formulation process on CLA uptake into SNE. See Example 4. [Figure 5A] Anti-6B IgG titers before immunization (pooled) and after the third dose (day 35) in mice immunized with the formulations listed in Table 4. Error bars represent the geometric mean of the 95% confidence interval. Transformed data analyzed by one-way ANOVA with Dunnett's post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ** **p<0.0001. NS is not significant. See Example 6. [Figure 5B] Table 4 shows the phagocytic and inactivating titers of serotype 6B opsonins in mice immunized with the formulations listed in Table 4, both before immunization (pooled) and after the third dose (day 35) (pooled). See Example 6. [Figure 6A] Ratio of serotype-specific IgG titers in adult rhesus monkeys after immunization with PCV24 containing CLA-SNE, compared to PCV24 containing APA, after the first dose (PD1: circle) and the second dose (PD2: square). PCV24 containing CLA-SNE (1200 μg / mL CLA-SNE) exhibits comparable or better immunogenicity compared to PCV24 containing APA. Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 7. [Figure 6B]Ratio of serotype-specific IgG titers in adult rhesus monkeys after immunization with the formulations listed in Table 5, compared to PCV24 containing APA, at post-dosage phase 2 (PD2). PCV24 containing CLA-SNE (round and triangular), CLA-LNP (square), or SNE (diamond) exhibits comparable or better immunogenicity compared to PCV24 containing APA. Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 7. [Figure 7A] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 1 and 3. See Example 7. [Figure 7B] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (individually / pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 4 and 5. See Example 7. [Figure 7C]Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 6A and 6B. See Example 7. [Figure 7D] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 6C and 7F. See Example 7. [Figure 7E] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 8 and 9V. See Example 7. [Figure 7F]Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 10A and 11A. See Example 7. [Figure 7G] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 12F and 14. See Example 7. [Figure 7H] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 15A and 15C. See Example 7. [Figure 7I]Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 18C and 19A. See Example 7. [Figure 7J] Table 5 shows the opsonin phagocytic killing titers in adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 19F and 22F. See Example 7. [Figure 7K] Table 5 shows the opsonin phagocytic killing titers of adult rhesus monkeys before immunization (pooled / individual), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 23B and 23F. See Example 7. [Figure 7L]Table 5 shows the opsonin phagocytic killing titers in adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotypes 24F and 33F. See Example 7. [Figure 7M] Table 5 shows the opsonin phagocytic killing titers in adult rhesus monkeys before immunization (pooled), after the first dose (day 14), and after the second dose (day 42) with the formulations listed. The administered dose volume was 0.1 mL per animal, resulting in CLA delivery doses of 0.08 mg and 0.12 mg for the PCV24 / CLA-SNE (80 μg) and PCV24 / CLA-SNE (120 μg) groups, respectively. Rhesus monkey serum was evaluated for functional antibodies determined by multiple opsonin phagocytosis assay (MOPA) for serotype 35B. See Example 7. [Figure 8A] Ratios of serotype-specific IgG titers in infant rhesus monkeys after immunization with PCV13, PCV24 containing CLA-LNP (120 μg dose), PCV24 containing CLA-SNE (295 μg CLA and 2.5 mg squalene), and PCV24 containing CLA-SNE (295 μg CLA and 0.5 mg squalene), compared to immunization with PCV24 containing APA, at 2nd dose (day 42). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 8. [Figure 8B] Ratio of serotype-specific IgG titers in infant rhesus monkeys after immunization with the selected formulations listed in Table 6, compared to PCV24 containing APA, at the time of the first dose (day 14). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 8. [Figure 8C]Ratio of serotype-specific IgG titers in infant rhesus monkeys after immunization with the selected formulations listed in Table 6, compared to PCV24 containing APA, at the second dose (day 42). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 8. [Figure 8D] Ratio of serotype-specific IgG titers in infant rhesus monkeys immunized with the selected formulations listed in Table 6, compared to PCV24 containing APA, at the third dose (day 70). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 8. [Figure 9] PCV24-immunized mice treated with the formulations (CLA-SNE, CLA-LNP, and SNE) are protected from intratracheal challenge of Streptococcus pneumoniae serotype 24F. See Example 9. [Figure 10A] Figures 10A-10D: Nanotracking (NTA) analysis of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 10A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 10B: SNE [40 mg / mL squalene], Figure 10C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], and Figure 10D: SNE [8 mg / mL squalene]). See Example 10. [Figure 10B] Figures 10A-10D: Nanotracking (NTA) analysis of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 10A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 10B: SNE [40 mg / mL squalene], Figure 10C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], and Figure 10D: SNE [8 mg / mL squalene]). See Example 10. [Figure 10C]Figures 10A-10D: Nanotracking (NTA) analysis of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 10A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 10B: SNE [40 mg / mL squalene], Figure 10C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], and Figure 10D: SNE [8 mg / mL squalene]). See Example 10. [Figure 10D] Figures 10A-10D: Nanotracking (NTA) analysis of CLA-SNE and SNE formulations stored at 4°C and 37°C for one month (Figure 10A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 10B: SNE [40 mg / mL squalene], Figure 10C: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], and Figure 10D: SNE [8 mg / mL squalene]). See Example 10. [Figure 11A] Figures 11A-11D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C (Figure 11A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 11B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], Figure 11C: SNE [40 mg / mL squalene], and Figure 11D: SNE [8 mg / mL squalene]). See Example 10. [Figure 11B] Figures 11A-11D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C (Figure 11A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 11B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], Figure 11C: SNE [40 mg / mL squalene], and Figure 11D: SNE [8 mg / mL squalene]). See Example 10. [Figure 11C]Figures 11A-11D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C (Figure 11A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 11B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], Figure 11C: SNE [40 mg / mL squalene], and Figure 11D: SNE [8 mg / mL squalene]). See Example 10. [Figure 11D] Figures 11A-11D: Dynamic light scattering (DLS) of CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C (Figure 11A: CLA-SNE [6 mg / mL CLA and 30 mg / mL squalene], Figure 11B: CLA-SNE [4 mg / mL CLA and 4 mg / mL squalene], Figure 11C: SNE [40 mg / mL squalene], and Figure 11D: SNE [8 mg / mL squalene]). See Example 10. [Figure 12A] CLA concentrations (mg / mL) measured by UPLC-CAD for CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C. See Example 11. [Figure 12B] Squalene concentrations (mg / mL) measured by UPLC-CAD for CLA-SNE and SNE formulations stored for 1 month at 4°C, 25°C, and 37°C. See Example 11. [Figure 13A] Serotype-specific stability of a pneumococcal polysaccharide-carrier protein conjugate co-production prepared using CLA-SNE (1.2 mg / mL CLA, 6.5 mg / mL squalene) and stored at 4°C for 1 month. See Example 12. [Figure 13B] Serotype-specific stability of a pneumococcal polysaccharide-carrier protein conjugate co-production prepared using CLA-SNE (1.2 mg / mL CLA, 1.2 mg / mL squalene) and stored at 4°C for up to 1 month. See Example 12. [Figure 13C]Serotype-specific stability of a pneumococcal polysaccharide-carrier protein conjugate co-production prepared using SNE (6.5 mg / mL squalene) and stored at 4°C for 1 month. See Example 12. [Figure 13D] Serotype-specific stability of a pneumococcal polysaccharide-carrier protein conjugate co-production prepared using SNE (0.4 mg / mL squalene) and stored at 4°C for 1 month. See Example 12. [Figure 14A] Ratio of serotype-specific IgG titers in adult rhesus monkeys after immunization with PCV21 containing CLA-SNE, compared to PCV21 (without adjuvant), at the 14th day after the first administration [referred to as D14PD1: square]. A 0.25 mL dose of PCV21 containing CLA-SNE (4 μg / mL per ST) yielded equivalent or better immunogenicity in D14PD1 compared to a 0.25 mL dose of PCV21 (4 μg / mL per ST). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 13. [Figure 14B] Ratio of serotype-specific IgG titers in adult rhesus monkeys after immunization with PCV21 containing CLA-SNE, compared to PCV21 (without adjuvant), at the first dose (day 28) [referred to as D28PD1: square]. A 0.25 mL dose of PCV21 containing CLA-SNE (4 μg / mL per ST) yielded equivalent or better immunogenicity in D28PD1 compared to a 0.25 mL dose of PCV21 (4 μg / mL per ST). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 13. [Figure 14C]Ratio of serotype-specific IgG titers in adult rhesus monkeys after immunization with PCV21 containing CLA-SNE, compared to PCV21 (without adjuvant), at the second dose (day 42) [referred to as D42PD2: square]. A 0.25 mL dose of PCV21 containing CLA-SNE (4 μg / mL per ST) yielded equivalent or better immunogenicity in D42PD2 compared to a 0.25 mL dose of PCV21 (4 μg / mL). Data for serotypes 6C and 15B are included to evaluate cross-reactivity. See Example 13. [Figure 15A] The CLA / squalene (w / w)% after dialysis is plotted against the "target" (w / w)% before self-assembly. The CLA / squalene w / w% ratio (X) was measured by reverse-phase ULC-CAD before and after self-assembly and nanoemulsion dialysis. See Example 14. [Figure 15B] The measured intensity-weighted Z-mean DLS diameter (X) of CLA-SNE nanoparticles after dialysis is plotted against the measured CLA / squalene (w / w)% after dialysis for each MNS formulation. See Example 14. [Figure 15C] The measured zeta potential of CLA-SNE squalene nanoparticles (X) after dialysis at pH 5.5 is plotted against the measured CLA / squalene (w / w)% after dialysis for each MNS preparation. See Example 14. [Figure 16] DLS Z-mean diameter of CLA-SNE samples treated with a formed aqueous phase (20 mM L-histidine) with gradually increasing pH. See Example 15. [Figure 17] Final [CLA] (mg / mL) of the CLA-SNE sample after treatment with the formed aqueous phase (20 mM L-histidine) with gradually increasing pH. See Example 15.

[0007] Detailed description of the invention Surprisingly, stable nanoemulsion (SNE) adjuvant formulations (cationic lipids) A pneumococcal conjugate composition containing (in the presence or absence of) aluminum adjuvant Pneumococcal conjugate composition containing adjuvant and / or lung adjuvant containing LNP adjuvant To produce an immunogenic response equivalent to or enhanced compared to Streptococcus conjugate compositions. It was discovered.

[0008] This invention relates to Streptococcus pneumoniae. umoniae) Polysaccharide-carrier protein conjugate and stable nanoemulsion (SNE) The present invention provides a pneumococcal conjugate composition containing ) and

[0009] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. A pneumococcal conjugate composition comprising t and SNE and a pharmaceutically acceptable carrier. provide.

[0010] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. To, emulsifiers and / or solubilizers and / or surfactants and, if desired, SNE containing cationic lipids or mixtures thereof, and pneumococcal conjugate A gate composition is provided.

[0011] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. A lung containing a surfactant and / or a terpene or mixture thereof. We provide a Streptococcus conjugate composition.

[0012] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. and surfactants and / or terpenes and / or cationic lipids or their The present invention provides a pneumococcal conjugate composition comprising a mixture of SNEs.

[0013] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. T, and one or more surfactants and one or more terpenes and one which may be used as desired. The present invention provides a pneumococcal conjugate composition comprising a cationic lipid-containing SNE.

[0014] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. It may be used with 1 to 3 surfactants and 1 to 3 terpenes as desired. The present invention provides a pneumococcal conjugate composition containing a SNE containing 1 to 3 cationic lipids. do.

[0015] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. It may also contain 1-2 surfactants and 1-2 terpenes, and may be used as desired. The present invention provides a pneumococcal conjugate composition comprising a SNE containing 1-2 cationic lipids. do.

[0016] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Centrioleart (SPAN-85), Polysorbate-20 (PS-20), or Poly Solvate-80 (PS-80), squalene, and catho, which may be used as desired. Contains glycemic lipids.

[0017] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Ntrioleart (SPAN-85), Polysorbate-20 (PS-20), Squalane It contains ions and cationic lipids, which may be used as desired.

[0018] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Centrioleart (SPAN-85), Polysorbate-20 (PS-20), or Poly Contains sorbate-80 (PS-80), squalene, and cationic lipids.

[0019] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Ntrioleart (SPAN-85), Polysorbate-20 (PS-20), Squalane Contains ions and cationic lipids.

[0020] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Centrioleart (SPAN-85), Polysorbate-20 (PS-20), or Poly Solvate-80 (PS-80), squalene, and cationic lipids (13Z, 16Z) Contains -N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine.

[0021] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. The present invention provides a pneumococcal conjugate composition comprising sorbita and SNE, wherein SNE is sorbita. Ntrioleart (SPAN-85), Polysorbate-20 (PS-20), Squalane N and cationic lipids (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-1 Contains 3,16-diene-1-amine.

[0022] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Lungs, and optionally, iv) lungs containing cationic lipids and SNEs We provide a Streptococcus conjugate vaccine. In some embodiments, the vaccine is a conjugate vaccine for Streptococcus pneumoniae. Jugate vaccines do not contain cationic lipids.

[0023] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squalene, and i which may be used if desired. v) Provide a pneumococcal conjugate vaccine containing a cationic lipid-containing SNE. In some embodiments, the pneumococcal conjugate vaccine contains cationic lipids. do not.

[0024] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Pneumococcal conjugate vaccine containing lenching agents and iv) SNE containing cationic lipids To provide.

[0025] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) squalene and iv) SN containing cationic lipids We provide a pneumococcal conjugate vaccine containing E.

[0026] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Len and iv)(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,1 We provide a pneumococcal conjugate vaccine containing SNE containing 6-dien-1-amine. ru.

[0027] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squalene and iv) (13Z,16Z)-N, Contains SNE containing N-dimethyl-3-nonyldocosa-13,16-diene-1-amine We provide a pneumococcal conjugate vaccine.

[0028] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Pneumococcal conjugate vaccine containing lenching agents and iv) SNE containing cationic lipids This provides a result in which the cationic lipids are not associated with lipid nanoparticles (LNPs).

[0029] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) squalene and iv) SN containing cationic lipids We provide a pneumococcal conjugate vaccine containing E, where cationic lipids are lipids It does not associate with LNPs.

[0030] The present invention further relates to the above-mentioned Streptococcus pneumoniae conjugate composition comprising a pharmaceutically acceptable carrier. provide.

[0031] In one embodiment, the composition comprises a cationic lipid CLA, CLX, or CLY. nothing.

[0032] In one embodiment, the composition comprises a cationic lipid CLA.

[0033] In one embodiment, the composition includes DLinDMA, DLinKC2DMA, and DL Selected from in-MC3-DMA, CLinDMA, and S-octylCLinDMA It contains cationic lipids.

[0034] In one embodiment, the Streptococcus pneumoniae polysaccharide in the composition - Each of the carrier protein conjugates is specific to Streptococcus pneumoniae. It contains serotype polysaccharides, where the polysaccharides in the conjugate are serotypes 1, 2, 3, and 4. 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20A, 2 0B, 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F and 38( One or more serums selected from any known serotype, including (but not limited to) these. Includes types. In another embodiment, the serotypes are 4, 6B, 9V, 14, 18C, 1 It includes, or is essentially derived from, or consists of, 9F and 23F. In one embodiment, the serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, Including 18C, 19A, 19F, and 23F, or essentially derived from them, or These consist of. In another embodiment, the serotypes are 1, 3, 4, 5, 6A, 6B, This includes 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, as well as It essentially becomes from them, or consists of them. In another embodiment, blood The clean type is 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, Including 15B, 18C, 19A, 19F, 22F, 23F and 33F, or those Essentially, they consist of, or comprise. In another embodiment, the serotype is 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 1 5B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B In another embodiment, In this case, the serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 1 2F, 14, 15A, de-O-acetylation - 15B, 18C, 19A, 19F, 22F, 2 This includes, or is essentially derived from, 3B, 23F, 24F, 33F, and 35B. or consists of them. In another embodiment, the serotypes are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19 A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, or including or Essentially consisting of or comprising. In another embodiment, the serotype is 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F , including 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B , or essentially become from them, or consist of them. In another embodiment The serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, and O-acetate. Chilled - 15B, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B, including, or essentially derived from, or derived from. In another embodiment, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11 A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23 B, 24F, 31, 33F and 35B, including, or essentially derived from them, It consists of those. In another embodiment, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 Including 3A, 23B, 24F, 31, 33F, and 35B, or essentially derived from them or consisting of them. In another embodiment, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17 Includes F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35B , or essentially become from them, or consist of them. In another embodiment The serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 1 6F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 3 5B includes, or essentially consists of, or comprises. Another implementation form In terms of state, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A. 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B, 24F, 31, 3 It includes, or is essentially derived from, or consists of 3F and 35B. In one embodiment, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12 F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20B, 22F, 2 Including 3A, 23B, 24F, 31, 33F, and 35B, or essentially derived from them or consisting of them. In another embodiment, the serotypes are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20B , including 22F, 23A, 23B, 24F, 31, 33F and 35B, or those To essentially become, or to consist of.

[0035] In one embodiment, the polysaccharide-carrier protein conjugate is serotype 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F , 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20(2 0A and 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35 Contains a polysaccharide selected from the group of pneumococcal serotypes consisting of F or 38. Another implementation In this state, the serotype group consists of 4, 6B, 9V, 14, 18C, 19F, and 23F. In another embodiment, the serotype group is 1, 3, 4, 5, 6A, 6B, 7F, It consists of 9V, 14, 18C, 19A, 19F and 23F. In another embodiment, The serotype groups are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, It consists of 19F, 22F, 23F and 33F. In another embodiment, the serotype The groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, It consists of 15B, 18C, 19A, 19F, 22F, 23F and 33F. Another fruit In terms of application methods, the serotype groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, and 10A. , 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, It consists of 23F, 24F, 33F and 35B. In another embodiment, the serotype The groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-acetylated - 15B, 18C, 19A, 19F, 22F, 23B, 23F , consists of 24F, 33F and 35B. In another embodiment, the group of serotypes is 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A , 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 3 It consists of 5B. In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N , 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20A, 22F , consisting of 23A, 23B, 24F, 31, 33F and 35B. Another embodiment is In this case, the serotype groups are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A, 20A, 22F, 23A, 23B , consisting of 24F, 31, 33F and 35B. In another embodiment, serotype The group consists of 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B In another embodiment, the serotype group consists of 3, 6A, 7F, 8, 9N, and 1. 0A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 23 It consists of A, 23B, 24F, 31, 33F, and 35B. In another embodiment The serotype groups are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, and O- Acetylated -15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F , 31, 33F and 35B. In another embodiment, the serotype group is 3 , 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, It consists of 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, and 35B. In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N, 10A, 11 A, 12F, 15A, 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23 It consists of B, 24F, 31, 33F and 35B. In another embodiment, serum The types are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, and de-O-acetylated. -15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B, 24F, 31 , 33F and 35B. In another embodiment, the serotype group is 3, 6A , 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A It consists of 20B, 22F, 23A, 23B, 24F, 31, 33F, and 35B.

[0036] In one embodiment, the present invention relates to seven different Streptococcus pneumoniae. Polysaccharide-carrier protein conjugate and the stable nanoemuls described herein The present invention provides a pneumococcal conjugate composition comprising any of the following: Treptococcus pneumoniae polysaccharide is found in serotypes 4, 6B, 9V, 14, 18C, and 19F. It consists of 23 floors.

[0037] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly The sugars are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F. It consists of 23 floors.

[0038] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly Sugar is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, It consists of floors 22, 23, and 33.

[0039] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly Sugar is serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 1 It consists of 4, 15B, 18C, 19A, 19F, 22F, 23F, and 33F.

[0040] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly The sugars consist of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 2 It consists of floors 4F, 33F, and 35B.

[0041] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly Sugar is serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 1 4, 15A, de-O-acetylation - 15B, 18C, 19A, 19F, 22F, 23B, 2 It consists of floors 3F, 24F, 33F, and 35B.

[0042] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly Sugar is serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 1 4, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33 It consists of F and 35B.

[0043] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly The sugars are serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, and 16. F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35 It consists of B.

[0044] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly The sugars are serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, and O-acetylated. -15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31 It consists of 33F and 35B.

[0045] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. A Nie polysaccharide-carrier protein conjugate and a SNE as described herein This provides a pneumococcus conjugate composition, wherein Streptococcus pneumoniae poly The sugars are serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, and 16. F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35 It consists of B.

[0046] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated (bonded), and the composition is sorbitan trioleate (SPAN-85) Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), It also contains squalene.

[0047] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further includes TO-80 (PS-80) and squalene.

[0048] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) The further comprises polysorbate-80 (PS-80) and squalene.

[0049] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), and It also contains squalene.

[0050] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains Bate-80 (PS-80) and squalene.

[0051] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), and further comprising squalene.

[0052] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains Bate-80 (PS-80) and squalene.

[0053] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), and further containing squalene.

[0054] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po It further contains resorbate-80 (PS-80) and squalene.

[0055] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), and further containing squalene.

[0056] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated, and the composition is sorbitan trioleate (SPAN-85), polysorbate Rubate-20 (PS-20) or Polysorbate-80 (PS-80), Squalene , and further comprising cationic lipids.

[0057] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further contains To-80 (PS-80), squalene, and cationic lipids.

[0058] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) It further contains polysorbate-80 (PS-80), squalene, and cationic lipids. nothing.

[0059] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), It further contains allenes and cationic lipids.

[0060] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipids.

[0061] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), squalene, and cationic lipids It also includes.

[0062] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipids.

[0063] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipids are further included.

[0064] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po It further contains resorbate-80 (PS-80), squalene, and cationic lipids.

[0065] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipids are further included.

[0066] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated, and the composition is sorbitan trioleate (SPAN-85), polysorbate Rubate-20 (PS-20) or Polysorbate-80 (PS-80), Squalene , and further comprising the cationic lipid CLA.

[0067] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further contains To-80 (PS-80), squalene, and cationic lipid CLA.

[0068] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) Alternatively, polysorbate-80 (PS-80), squalene, and cationic lipid CLA It also includes.

[0069] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), The material further contains allene and the cationic lipid CLA.

[0070] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipid CLA.

[0071] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), squalene, and cationic lipid C This also includes LA.

[0072] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipid CLA.

[0073] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipid CLA further comprise.

[0074] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po The following further contains resorbate-80 (PS-80), squalene, and cationic lipid CLA. nothing.

[0075] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipid CLA further comprise.

[0076] In one embodiment of the composition, SNE includes PS-20. In another embodiment, the SNE includes the PS-80.

[0077] In one embodiment of the composition, the composition is any other streptococcus. It does not contain polysaccharide-carrier protein conjugates containing the S. pneumoniae serotype polysaccharide. stomach.

[0078] In one embodiment of the composition of the present invention, the carrier protein is OMPC, PhtD pLys, DT (diphtheria toxoid), TT (tetanus toxoid), TT fragment C The other is selected from pertussis toxoid, cholera toxoid and CRM197. In this embodiment, the carrier protein is CRM197.

[0079] This invention relates to 5 mM to 40 mM histidine and 25 mM histidine at pH 5.1 to 7.0. The present invention provides the composition further comprising ~300 mM NaCl.

[0080] This invention relates to approximately 20 mM histidine and approximately 75 mM NaCl at a pH of approximately 5.8. The present invention provides the composition further comprising the above.

[0081] The present invention further provides the above composition comprising 20 mM histidine and 75 mM NaCl at pH 5.8 .

[0082] The present invention further provides the above composition comprising 5 mM to 40 mM histidine, 0.0125 % to 0.2% PS-20 or PS-80, and 25 mM to 300 mM NaCl at pH 5.1 to 7.0 .

[0083] The present invention further provides the above composition comprising about 20 mM histidine, 0.05% PS-20 or PS-80, and about 75 mM NaCl at about pH 5.8.

[0084] The present invention further provides the above composition comprising 20 mM histidine, 0.05% PS-20 or PS-80, and 75 mM NaCl at pH 5.8.

[0085] The present invention also provides a method for preparing a pneumococcal conjugate composition, which comprises: 1) a Streptococcus pneumoniae polysaccharide-carrier protein conju gate; and 2) an SNE comprising i) sorbitan trioleate (SPAN-85), (ii) polysorbe ate 20 (PS-20) or polysorbate 80 (PS-80), iii) squal ene, and optionally iv) a cationic lipid. .

[0086] The present invention also provides a method for preparing a pneumococcal conjugate composition, which comprises: 1) a Streptococcus pneumoniae polysaccharide-carrier protein conju gate; and 2) an SNE comprising i) sorbitan trioleate (SPAN-85), (ii) polysorbe ate 20 (PS-20), iii) squalene, and optionally i v) a cationic lipid. To provide.

[0087] This invention also relates to the Streptococcus pneumoniae polysaccharide-carrier protein conjugate. The present invention provides a method for producing a pneumococcal conjugate composition containing Streptococcus pneumoniae and SNE.

[0088] The present invention also relates to the treatment of pneumococcal disease using the pneumococcal conjugate composition of the present invention. To provide a treatment or preventive measure.

[0089] The present invention also relates to the pneumococcal conjugate of the present invention for the treatment or prevention of pneumococcal disease. The use of the composition is provided.

[0090] definition The singular form used throughout this specification and in the appended claims is used only when clearly indicated in context. It includes multiple objects, as long as they do not contradict each other.

[0091] The following abbreviations apply throughout this specification and the appended claims: APA aluminum phosphate adjuvant CLA ((13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,1 6-diene-1-amine) DMSO (Dimethyl Sulfoxide) ECL Electrochemiluminescence GMT Geometric Mean Titer HPSEC High-Performance Size Exclusion Chromatography ID intradermal IM Muscle LNP lipid nanoparticles LOS Lipooligosaccharide LPS (Lipopolysaccharide) ME Microemulsion MNS Microfluidic Nanoemulsion Self-Assembly Mw Molecular weight NE Nanoemulsion NMWCO Nominal Molecular Weight Cutoff OPA Opsonin Phagocytosis Assay PCV Pneumococcal Conjugate Vaccine PD1 After first administration PD2 After second administration PD3 After third administration PHE Pre-homogenization Emulsion PnPs Pneumococcal Polysaccharide Ps Polysaccharide PS-20 Polysorbate-20 PS-80 Polysorbate-80 SNE Stable Nanoemulsion SPAN-85 Sorbitan Trioleate ST6B or ST-6B Serotype 6B w / v Weight per volume

[0092] As used herein with respect to a value, the term "about" refers to a value that is the same as the reference value, or a value that is contextually similar to the reference value. In general, a person skilled in the art who is familiar with the situation (context) will understand the absolute amount and / or relative degree of variation encompassed by "about" in that situation. For example, in some embodiments, the term "about" can encompass a range within 25% of the reference value, within 20%, within 19%, within 18%, within 17%, within 16%, within 15%, within 1 4%, within 13%, within 12%, within 11%, within 10%, within 9%, within 8%, within 7 %, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less than 1% of the reference value.

[0093] As used herein, the term "alkenyl" refers to a straight-chain, cyclic or branched unsaturated aliphatic hydrocarbon having the specified number of carbon atoms. In one embodiment, the alkenyl group contains 8 to 24 carbon atoms (C8-C 24Alkenil). 1 In one embodiment, the alkenyl group is linear. In another embodiment, The alkenyl group is branched. In another embodiment, the alkenyl group is substituted. I haven't done that.

[0094] As used herein, the term "alkyl" refers to a linear chain having a specified number of carbon atoms. This refers to cyclic, cyclic, or branched saturated aliphatic hydrocarbons. In one embodiment, A The lukyl group contains 8 to 24 carbon atoms (C8-C 24 Alkyl). One embodiment In this embodiment, the alkyl group is linear. In another embodiment, the alkyl group is It is branched. In another embodiment, the alkyl group is not substituted.

[0095] The term "adjuvant" as defined herein enhances the immunogenicity of the immunogenic composition of the present invention. Substances that help to strengthen (for example, increase, accelerate, extend, or regulate) As disclosed herein, SNE can be used as an adjuvant according to the present invention. It should be used. Adjuvants are weakly immunogenic when administered alone (for example) (i.e., an immune response to an antigen that does not induce antibody titer or cellular immune response at all or only weakly) This enhances the immune response, increases antibody titers against the antigen, and / or achieves an immune response in the individual. This may reduce the effective dose of antigen. When used in this specification, "adjuvant "Added composition" is a composition containing an adjuvant.

[0096] As used herein, the term "administration" refers to providing an active substance, composition, or preparation. It means the act of administering to the human body. Examples of routes of administration to the human body include the eyes, mouth, and skin. , nose (intranasal cavity), lungs (inhalation), rectum, vagina, oral mucosa (cheek), ear, injection [e.g., intravenous ( IV) Subcutaneous, intratumoral, intraperitoneal, intramuscular (IM), intradermal (ID), etc. It is possible.

[0097] The term "antigen" refers to any antigen that can elicit one or more immune responses. Proteins (including recombinant proteins), polypeptides or peptides (including synthetic peptides) It may be (m). In certain embodiments, the antigen is a lipid or a carbohydrate (polysaccharide). In certain embodiments, the antigen is a protein extract, a cell (including tumor cells) or It is a tissue. Antigens are antigens that elicit humoral immune responses and / or CTL immune responses. It is possible. The antigen of the present invention is Streptococcus pneumoniae polysaccharide.

[0098] The term "cationic lipid" refers to a lipid that has a net positive charge at a selected pH, such as physiological pH. This refers to lipid species that have a charge. Cationic lipids are used in multi-component SNE adjuvant formulations. It can be used as an ingredient. Cationic lipids include those disclosed in the following (and Those skilled in the art will recognize that this may include (but is not limited to): U.S. Patent Publication Application publication number US2008 / 0085870, US2008 / 0057080, US200 9 / 0263407, US2009 / 0285881, US2010 / 0055168, US2010 / 0055169, US2010 / 0063135, US2010 / 007 6055, US2010 / 0099738, US2010 / 0104629, US201 3 / 0017239 and US2016 / 0361411, International Patent Application Publication Number WO201 1 / 022460, WO2012 / 040184, WO2011 / 076807, WO2 010 / 021865, WO2009 / 132131, WO2010 / 042877, W O2010 / 146740 and WO2010 / 105209, and U.S. Patent No. U S5,208,036, US5,264,618, US5,279,833, US5,2 83,185, US6,890,557, and US9,669,097.

[0099] As used herein, the term “composition” refers to an active pharmaceutical or biological component (e.g., , pneumococcal polysaccharide-carrier protein conjugate and SNE), one or more additional components It refers to a preparation containing the same amount of ingredients. The term "composition" is used in conjunction with "pharmaceutical composition" and "preparation." They are used interchangeably. The composition can be liquid or solid (e.g., freeze-dried). Depending on the circumstances, additional ingredients may be included, such as pharmaceutically acceptable excipients, additives, diluents, and batters. Fat, sugar, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, freeze-dried Lyo-protectant, solubilizer, emulsifier, salt, adjuvant, tincture It contains a performance enhancer, a transport (delivery) vehicle, and an antimicrobial preservative. The composition is used It is nontoxic to the subject at the dose and concentration.

[0100] When used in reference to the compositions of the present invention, the word "includes" means adjuvants and excipients. Inclusion of any other component such as an agent, or one or more polysaccharide-carriers not specifically mentioned. This refers to the addition of protein conjugates.

[0101] When used in reference to polyvalent polysaccharide-carrier protein conjugate preparations, the term "consists of" Or the word "karanari" refers to those specific Streptococcus pneumoniae polysaccharides - Other Streptococcus species containing carrier protein conjugates and derived from different serotypes. This relates to a formulation that does not contain S. pneumonier polysaccharide-carrier protein conjugate. be.

[0102] "From to substantial" and its derivatives, for example, "from substantial" or "from substantial" "Qualitatively" means the inclusion of any element or group of elements listed, and the relationship between the listed elements and the type of elements. Other elements of similar or different nature that are identified as part of a specific administration regimen, method, or combination The discretionary inclusion of elements that do not substantially alter the fundamental or novel properties of the product (i.e., inclusion) This indicates whether or not it is included.

[0103] The terms "de-O-acetylated-15B" or "de-O-acetylated-15B" as used herein Alternatively, "de-O-Ac-15B" refers to a de-O-Ac-15B that has an O-acetyl content of less than 5% per repeating unit. This refers to O-acetylated serotype 15B. In another embodiment, it refers to O-acetylated serotype 15B. The content is less than 1% per repeating unit. In another embodiment, O-acetyl content The amount is less than 0.5% per repeating unit. In another embodiment, O-acetyl The content is less than 0.1% per repeating unit. Methods for de-O-acetylation are described in the technical field. This is publicly known, for example, Rajam et al., Clinical and Vaccine I This is described in mmunology, 2007, 14(9):1223-1227.

[0104] As used herein, the term "dosage" refers to the amount taken or consumed at a specific time. This refers to the amount of a substance, API (active pharmaceutical ingredient), formulation, or composition that is recommended to be used.

[0105] In this specification, the terms "immunogenic" or "immunogenic" refer to the antigen being targeted. This means that it can induce an immune response. The term "immunogenic composition" means that in the subject, This refers to a substance, API, formulation, or composition that can induce an immune response. The denjugate composition is an immunogenic composition.

[0106] Those who "require treatment" have previously been exposed to or infected with Streptococcus pneumoniae. Those who have done so, those who have previously been vaccinated against Streptococcus pneumoniae, and Individuals who are susceptible to infection, or any individual for whom a reduction in the likelihood of infection is desirable, such as immunocompromised individuals, high risk of infection This includes elderly individuals, children, adults, or healthy individuals.

[0107] The phrase "suitable for the prevention of pneumococcal disease" refers to pneumococcal disease in general, pneumococcal pneumonia, and lung disease. Streptococcal meningitis, pneumococcal bacteremia, caused by Streptococcus pneumoniae. Invasive diseases and otitis media caused by Streptococcus pneumoniae ( Any blood of Streptococcus pneumoniae (not limited to these) Regarding the prevention of one or more diseases caused by a clear strain, the vaccine or composition is used in the U.S. Dietary The U.S. Food and Drug Administration This means that it is approved by one or more regulatory authorities.

[0108] As used herein, the term "lipid" refers to a large number of substances that are insoluble in water or have low water solubility. Organic fatty acid esters are characterized by their solubility in organic solvents. This refers to any of the group of compounds. Lipids can be classified into at least three classes. (1) "Simple lipids": This includes fats and oils and waxes; (2) "Complex lipids": This includes, for example, phospholipids and glycolipids; as well as (3) "Derivatives" "Lipids": This includes, for example, steroids.

[0109] As used herein, the term "lipid nanoparticles" (or "LNP") refers to multiple classes. Lipids of the name and / or type, with dimensions of 10 to 1000 nanometers in length or width ( For example, it means a lipid composition that forms particles having a maximum length or width. For example, according to the present invention, lipid nanoparticles are not composed solely of cationic lipids.

[0110] As used herein, the term "neutral lipid" refers to a lipid that is uncharged or neutral at a selected pH. This refers to lipid species that exist in an amphoteric form. In physiological pH, such lipids... For example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, Lamid, sphingomyelin, cephalin, cholesterol, cerebroside and diacin It contains glycerol.

[0111] "Patient" (or, as referred to herein, "subject") is a patient with streptococcal disease. This refers to mammals that can be infected with Cass pneumoniae. In a preferred embodiment, the affected The person is a human being. Patients can be treated prophylactically or therapeutically. Prophylactic treatment is for pneumococcal disease. Sufficient prevention to reduce the likelihood or severity of the disease or its effects, such as pneumococcal pneumonia. To provide immunity. Severity of Streptococcus pneumoniae infection or its clinical effects. Therapeutic treatment may be administered to reduce or prevent recurrence. Preventive treatment is also available. As described in the details, the present invention is carried out using the pneumococcal conjugate composition. The pneumococcal conjugate composition or vaccine of the present invention is intended for use in the general population or in pneumonia. Individuals at high risk of cocci infection, such as the elderly, or those living with or caring for the elderly. It can be administered to those who are undergoing [a certain condition].

[0112] As used herein, "PCV1" is conjugated (bound) to a carrier protein. A single streptococcus containing a capsular polysaccharide derived from the serotype of Streptococcus pneumoniae Monovalent pneumococcus conjugate containing Cass pneumoniae polysaccharide-carrier protein conjugate This means a vaccine or composition. In certain embodiments, the carrier protein is C It's RM197.

[0113] As used herein, "PCV13" refers to a stoconjugated stoconjugate to a carrier protein. Each of the 13 streptococci contains a capsular polysaccharide derived from the serotype of Leptococcus pneumoniae. 13-valent Streptococcus pneumoniae containing Tococcus pneumoniae polysaccharide-carrier protein conjugate This means a vaccine or composition, where Streptococcus pneumoniae The serotypes of E are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, and 19F. and 23F. In certain embodiments, Streptococcus pneumoniae poly Each of the carrier proteins in the sugar-carrier protein conjugate is CRM197.

[0114] As used herein, "PCV21" refers to a carrier protein that, when used herein, Each of the denjugate-derived capsular polysaccharides from the serotype of Streptococcus pneumoniae This contains 20 Streptococcus pneumoniae polysaccharide-carrier protein conjugates. This means a 21-valent pneumococcal conjugate vaccine or composition containing, where, The serotypes of *Ptococcus pneumoniae* are 3, 6A, 7F, 8, 9N, 10A, 11A, and 1 2F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31 , 33F and 35B, as well as the following 15 serotypes, namely 15B, 15C This is one of the de-o-acetylated -15B. In certain embodiments, serum group 15 blood The clear form is serotype 15C or de-O-acetylated-15B. In another embodiment, In this case, serotype 15 is serotype de-O-acetylated -15B. In this case, 1 of the Streptococcus pneumoniae polysaccharide-carrier protein conjugates The carrier protein shown above is CRM197. In other embodiments, streptococcus Each of the carrier proteins in the Cus pneumoniae polysaccharide-carrier protein conjugate The quality is CRM197.

[0115] As used herein, "PCV24" refers to a carrier protein that, when used herein, Each of the denjugate-derived capsular polysaccharides from the serotype of Streptococcus pneumoniae This contains 23 Streptococcus pneumoniae polysaccharide-carrier protein conjugates. This means a 24-valent pneumococcal conjugate vaccine or composition containing, where, The serotypes of Ptococcus pneumoniae are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V. 10A, 11A, 12F, 14, 15A, 18C, 19A, 19F, 22F, 23B, 2 3F, 24F, 33F and 35B, as well as the following 15 serotypes, i.e., 15 It is one of B, 15C, or de-O-acetylated-15B. In certain embodiments, Serogroup 15 serotype is serotype 15C or deoxy-acetylated -15B. In terms of application, serotype 15 is serotype de-O-acetylated -15B. In one embodiment, the Streptococcus pneumoniae polysaccharide-carrier protein conjugate One or more of the gate's carrier proteins are CRM197. In other embodiments, Each of the Streptococcus pneumoniae polysaccharide-carrier protein conjugates, The carrier protein is CRM197.

[0116] With respect to carriers, diluents, or excipients of pharmaceutical compositions, the phrase "pharmaceutically acceptable" means: The carrier, diluent, or excipient is compatible with the other components of the composition and is not harmful to the recipient. This indicates that it is necessary that it is not the case.

[0117] The terms "pneumococcal conjugate" or "pneumococcal polysaccharide-carrier protein" used herein The term "protein conjugate" refers to the polysaccharide-carrier protein conjugate of Streptococcus pneumoniae. It means "jugate".

[0118] The term “pneumococcal conjugate vaccine” (or “PCV”) as used herein This refers to a disease or pathology caused by a serotype of Streptococcus pneumoniae. Contains a pneumococcal polysaccharide-carrier protein conjugate that provides active immunity against the disease state. It is a pharmaceutical preparation or composition.

[0119] As used herein, the terms "stable nanoemulsion" or "SNE" refer to Streptococcus pneumoniae. Emulsifiers and / or solubilizers having adjuvant properties in adjugate vaccines It means a formulation of surfactants and / or lipids. In particular, SNE means 1) 1) Sorbitan Triolet (SPAN-85), 2) Polysorbate-20 (PS-20) ), 3) squalene, and optionally 4) SN containing cationic lipids This refers to an E-adjuvant preparation.

[0120] "Surfactants" are stabilizing components in multi-component SNE adjuvant formulations, and the following: This includes: polyoxyethylene sorbitan ester surfactants (generally Tween, (especially referred to as PS-20 and PS-80), ethylene oxide (EO), propylene A copolymer of oxide (PO) and / or butylene oxide (BO) and DOW Products sold under the trademark name FAX, for example, linear EO / PO block copolymer. Mer (poloxamer); with varying numbers of repeating ethoxy (oxy-1,2-ethanediyl) groups. Possible octoxynol [octoxynol-9 (Triton X-100 or t -Octylphenoxypolyethoxyethanol) is of particular interest]; (Octylph Ethoxyethanol (IGEPAL CA-630 / NP-40); Noni Luphenol ethoxylate, e.g., Tergitol® NP series; lauryl Polyoxyethylene fats derived from cetyl, stearyl, and oleyl alcohols Ethers (known as Brij surfactants), such as triethylene glycol mono Lauryl ether (Brij30); and sorbitan ester (generally referred to as SPAN) (and this is publicly known), for example, sorbitan triolet (Span-85, Tween-85) Or [2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadeca-9 -Enoyl]oxyoxolan-2-yl]-2-[(Z)-octadeca-9-enoyl Oxyethyl (Z)-octadeca-9-enoate and sorbitan monolaurate In one embodiment, the surfactant is a sorbitan ester and a poloxamer. Yes. In one embodiment, the surfactant is polysorbate-20 (PS-20). It is also known as polysorbate-80 (PS-80).

[0121] "Terpenes" are stabilizing components in multi-component SNE adjuvant formulations, and include the following: (Not limited to these) includes: monoterpenes, e.g., geraniol, Terpineol, limonene, myrcene, linalool, and pinene; sesquiterpenes, e.g. For example, humulenes, farnesenes, and farnesols; diterpenes, such as cafestol. , kahweol, semblen and taxadiene; triterpenes, such as squalene and squalante; tetraterpenes, such as acyclic lycopene, monocyclic gamma-carotene, Bicyclic alpha- and beta-carotenes; polyterpines and norisopredoids. In this embodiment, the terpene is squalene.

[0122] The term "therapeutic effective dose" refers to the amount of a person or animal that produces the desired therapeutic effect. A suitable composition or vaccine quantity, for example, to induce an immune response and treat a disease or its symptoms. To cure, prevent, or suppress the onset and progression of a disease or its symptoms The required amount, and / or the amount necessary to improve the symptoms of the disease or cause the disease to regress. It means the required amount. A person skilled in the art can easily determine the therapeutically effective amount of a given composition or vaccine. Shut up.

[0123] As used herein, the term "value" refers to the number of specified polysaccharide-carrier tans in the composition. This indicates the presence of protein conjugates.

[0124] As used herein, the terms "vaccine" or "vaccine composition" refer to a substance that stimulates antibody production. This refers to biological agents used to intensify and provide immunity against infectious diseases.

[0125] Cationic lipids Cationic lipids and methods for producing cationic lipids are well known in the art.

[0126] In some embodiments, the cationic lipid is any of the following: Contains cationic lipids: U.S. Patent Application Publication No. US 2008 / 0085870, US 2008 / 0057080, US 2009 / 0263407, US 2009 / 0285881, US 2010 / 0055168, US 2010 / 0055169, US 2010 / 0063135, US 2010 / 0076055, US 2010 / 0099738, US 2010 / 0104629, US 2013 / 0017239 US 2016 / 0361411, International Patent Application Publication Number WO2011 / 02246 0 A1;WO2012 / 040184, WO2011 / 076807, WO2010 / 021865, WO2009 / 132131, WO2010 / 042877, WO201 0 / 146740, WO2010 / 105209, and U.S. 5,208,03 No. 6, No. 5,264,618, No. 5,279,833, No. 5,283,185, No. Numbers 6,890,557 and 9,669,097.

[0127] In some embodiments, the cationic lipid of the present invention is represented by the following formula 1: [ka] (In the formula, R 1 and R 2 Each of them is methyl; R 3 H is; n is either 1 or 2; L1 is C8-C 24 Alkyl and C8-C 24 Selected from alkenil; and L2 is selected from C4-C9 alkyl and C4-C9 alkenyl. or having a structure represented by any pharmaceutically acceptable salt or stereoisomer thereof. ru.

[0128] Cationic lipids can be aminoalkyl lipids.

[0129] Cationic lipids can be asymmetric aminoalkyl lipids.

[0130] In one embodiment of the present invention, the cationic lipid is (13Z,16Z)-N,N- Dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA), or (6 Z,9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,2 9-tetraen-18-amine (CLX), or N,N-dimethyl-1-((1S,2 It is R)-2-octylcyclopropyl)heptadecane-8-amine (CLY).

[0131] In another embodiment of the present invention, cationic lipids are selected from the following: : DLinDMA; DLinKC2DMA; DLin-MC3-DMA; CLinDMA; S-octyl clinDMA; (2S)-1-{7-[(3P)-cholesta-5-en-3-yloxy]heptyloxy cy-3-[(4Z)-deca-4-en-1-yloxy]-N,N-dimethylpropane -2-amine; (2R)-1-{4-[(3P)-cholesta-5-en-3-yloxy]butoxy}- 3-[(4Z)-deca-4-en-1-yloxy]-N,N-dimethylpropane-2- Amine; 1-[(2R)-1-{4-[(3β)-cholesta-5-en-3-yloxy]butoxy C-3-(octyloxy)propane-2-yl]guanidine; 1-[(2R)-1-{7-[(3β)-cholesta-5-en-3-yloxy]hepti Luoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-die [n-1-yloxy]propan-2-amine; 1-[(2R)-1-{4-[(3β)-cholesta-5-en-3-yloxy]butoxy cy-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1 -yloxy]propan-2-amine; (2S)-1-({6-[(3P))-cholesta-5-en-3-yloxy]hexyl }oxy)-N,N-dimethyl-3-[(9Z)-octadeca-9-en-1-yloxy [C]propan-2-amine; (3β)-3-[6-{[(2S)-3-[(9Z)-Octadeca-9-en-1-yl Oxyl]-2-(pyrrolidine-1-yl)propyl]oxy}hexyl)oxy]cholesterol Star-5-End; (2R)-1-{4-[(3P)-cholesta-5-en-3-yloxy]butoxy}- 3-(octyloxy)propan-2-amine; (2R)-1-({8-[(3P)-Cholesta-5-en-3-yloxy]octyl} Oxy)-N,N-dimethyl-3-(pentyloxy)propane-2-amine; (2R)-1-({8-[(3P)-Cholesta-5-en-3-yloxy]octyl} Oxy)-3-(heptyloxy)-N,N-dimethylpropane-2-amine; (2R)-1-({8-[(3P)-Cholesta-5-en-3-yloxy]octyl} Oxy)-N,N-dimethyl-3-[(2Z)-penta-2-en-1-yloxy] Ropan-2-amine; (2S)-1-Butoxy-3-({8-[(3P)-Colesta-5-En-3-Iluoki [C]octyl}oxy)-N,N-dimethylpropane-2-amine; (2S-1-({8-[(3P)-Cholesta-5-En-3-Iloxy]Octyl}O Kishi)-3-[2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-he Xadecafluorononyl)oxy]-N,N-dimethylpropane-2-amine; 2-amino-2-{[(9Z,12Z)-octadeca-9,12-diene-1-ylox [C]methyl}propane-1,3-diol; 2-amino-3-({9-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest -5-en-3-yloxy]nonyl}oxy)-2-{[(9Z,12Z)-octade [9,12-diene-1-yloxy]methyl}propan-1-ol; 2-amino-3-({6-[(3β,8ξ,9ξ,14ξ,17ξ,20ξ)-cholest -5-en-3-yloxy]nonyl}oxy)-2-{[(9Z)-octadeca-9- En-1-yloxy]methyl}propan-1-ol; (20Z,23Z)-N,N-dimethylnonacosa-20,23-diene-10-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-9-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-8-amine; (13Z,16Z)-N,N-dimethyldocosa-13,16-diene-5-amine; (12Z,15Z)-N,N-dimethylhenicotha-12,15-diene-4-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-6-amine; (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-7-amine; (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine ; (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine; (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-9-amine; (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-8-amine; (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine; (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine; (22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10- Amine; (21Z,24Z)-N,N-dimethyltriaconta-21,24-diene-9-amine ; (18Z)-N,N-dimethylheptacosa-18-ene-10-amine; (17Z)-N,N-dimethylhexacosa-17-ene-9-amine; (19Z,22Z)-N,N-dimethyloctacosa-19,22-diene-7-amine; N,N-dimethylheptacosan-10-amine; (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-diene-10-a Min; 1-[(11Z,14Z)-1-nonylcosa-11,14-diene-1-yl]pyrrolidi hmm; (20Z)-N,N-dimethylheptacosa-20-ene-10-amine; (15Z)-N,N-dimethylheptacosa-15-ene-10-amine; (14Z)-N,N-dimethylnonacosa-14-ene-10-amine; (17Z)-N,N-dimethylnonacosa-17-ene-10-amine; (24Z)-N,N-dimethyltriaconto-24-ene-10-amine; (20Z)-N,N-dimethylnonacosa-20-ene-10-amine; (22Z)-N,N-dimethylhenthriaconta-22-ene-10-amine; (16Z)-N,N-dimethylpentacosa-16-ene-8-amine; (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-diene-1 -amine; (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1- Amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane -8-amine; 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane- 10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane- 10-amine; N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan -10-amine; N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycline Ropropylmethylcyclopropylnonadecan-10-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane -8-amine; N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradeca n-5-amine; N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl Ludodecane-1-amine; 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane -9-amine; 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane- 6-amine; N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane -8-amine; and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene -10-amine; or pharmaceutically acceptable salts thereof, or any stereoisomer of the foregoing.

[0132] In another embodiment of the present invention, the cationic lipid is (13Z,16Z)-N N-dimethyl-3-nonyldocosa-13,16-dien-1-amine or its pharmaceutical Selected from the acceptable salts or stereoisomers.

[0133] In another embodiment of the present invention, the cationic lipid is (13Z,16Z)-N It is N-dimethyl-3-nonyldocosa-13,16-diene-1-amine (CLA). .

[0134] This disclosure, in particular, concerns pneumococcal conjugates and three SNE components, namely, 1) 1) Sorbitan Triolet (SPAN-85), 2) Polysorbate-20 (PS-20) ) or polysorbate-80 (PS-80), and 3) a composition comprising squalene provide.

[0135] In some embodiments, SNE is 32-97 mol% squalene, 1-34 mol Contains % SPAN-85 and 1-34 mol% PS-20 or PS-80.

[0136] In some embodiments, SNE is 86-98 mol% squalene, 1-7 mol% It contains SPAN-85 and 1-7 mol% of PS-20 or PS-80.

[0137] In some embodiments, SNE is 92-94 mol% squalene, 3-4 mol% It contains SPAN-85 and 3-4 mol% of PS-20 or PS-80.

[0138] In one embodiment of the present invention, SNE is 92.91 mol% squalene, 3.9 Contains 8 mol% SPAN-85 and 3.11 mol% PS-20 or PS-80 .

[0139] This disclosure, in particular, concerns pneumococcal conjugates and four SNE components, namely, 1) 1) Cationic lipids, 2) Sorbitan trioleate (SPAN-85), 3) Polysorbate TO-20 (PS-20) or polysorbate-80 (PS-80), and 4) Squa The present invention provides compositions containing len. Specific SNE compositions include cationic lipids (13Z, 16Z )-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine("CLA " or "CLA-SNE" if cationic lipids are included in the SNE.

[0140] In some embodiments, SNE is 1-60 mol% cationic lipid, 32-97 mol% squalene, 1-4 mol% SPAN-85, and 1-4 mol% PS-20 or including PS-80.

[0141] In some embodiments, SNE is 10-14 mol% cationic lipid, 78-8 4 mol% squalene, 1-6 mol% SPAN-85, and 1-6 mol% PS-2 Includes 0 or PS-80.

[0142] In some embodiments, SNE is 40-46 mol% cationic lipid, 44-5 2 mol% squalene, 1-8 mol% SPAN-85, and 1-8 mol% PS-2 Includes 0 or PS-80.

[0143] In one embodiment of the present invention, SNE is 13.82 mol% cationic lipid, 8 0.07 mol% squalene, 3.43 mol% SPAN-85, and 2.68 mol% This includes the PS-20 or PS-80.

[0144] In one embodiment of the present invention, SNE is 44.5 mol% cationic lipid, 51 0.56 mol% squalene, 2.21 mol% SPAN-85, and 1.72 mol% Includes PS-20 or PS-80.

[0145] In some embodiments of the present invention, SNE is a surfactant, a mixture of multiple surfactants Select from compound, phospholipid, terpene, terpenoid, triterpene, or combination thereof. It further contains one or more noncationic lipids that may be affected.

[0146] In some embodiments, surfactants may include (but are not limited to) the following: (Not something that is done): Polyoxyethylene sorbitan ester surfactant (generally Tw (referred to as een), especially PS-20 and PS-80; ethylene oxide (EO), p A copolymer of propylene oxide (PO) and / or butylene oxide (BO) Products sold under the product name DOWFAX (trademark), such as linear EO / PO blocks Goji copolymer; the number of repeating ethoxy(oxy-1,2-ethanediyl) groups can vary. Octoxynol [Octoxynol-9 (Triton X-100 or t-octoxynol)] There is particular interest in (octylphenoxypolyethoxyethanol); ) Polyethoxyethanol (IGEPAL CA-630 / NP-40); Nonylpheno Ethoxylate, e.g., Tergitol® NP series; lauryl, cetyl , polyoxyethylene fatty ethers derived from stearyl and oleyl alcohols (known as a Brij surfactant), for example, triethylene glycol monolauryl Ether (Brij30); and sorbitan ester (commonly known as SPAN) (There is) For example, sorbitan triolet (Span-85, Tween-85 or [ 2-[(2R,3S,4R)-4-hydroxy-3-[(Z)-octadeca-9-enoy [Oxyoxolan-2-yl]-2-[(Z)-Octadeca-9-enoyl]oxy Ethyl (Z)-octadeca-9-enoate) and sorbitan monolaurate.

[0147] In some embodiments, a mixture of surfactants, for example, PS-20 / Span85 Alternatively, a PS-80 / Span85 mixture may be used. Polyoxyethylene sorbitan Esters, for example, polyoxyethylene sorbitan monooleate (PS-80), and oc Toxinol, for example, t-octylphenoxypolyethoxyethanol (Triton X-1 The combination with 00) is also suitable. Another useful combination is laureth 9 and polyoxyen. Contains ethylene sorbitan ester and / or octoxynol.

[0148] In some embodiments, the preferred amount of surfactant or emulsifier is as follows: Yes: Polyoxyethylene sorbitan ester (e.g., PS-20 or PS-80) 0.01–10 mol%, especially about 1–4 mol%; octyl or nonylphenoxypolyoxy Cethanol (e.g., Triton X-100, or other interfaces in the Triton series) Activator) 0.001-10 mol%, especially about 1-4 mol% w / v, especially 0.01-0.1 % w / v; polyoxyethylene ether (e.g., laureth-9) 0.1-20 mol%, Preferably 0.5 to 10 mol%, particularly 1 to 4% mol%, or about 10% by mass.

[0149] In some embodiments, the phospholipid is a natural phospholipid, such as phosphatidyl phosphate. Phosphorus (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol PG, phosphatidylserine (PS), phosphatidylinositol (PI), Phosphatidic acid (phosphatidate) (PA), dipalmitoylphosphatidylcholine Monoacyl-phosphatidylcholine (LisoPC), 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphocholine (POPC), N-acyl-PE, phosphoinosity Phosphorus may include, but is not limited to, phosphorus and phosphosphingolipids. Lipid derivatives include phosphatidic acid (DMPA, DPPA, DSPA) and phosphatidyl Corin (DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, D EPC), phosphatidylglycerol (DMPG, DPPG, DSPG, POPG), Phosphatidylethanolamine (DMPE, DPPE, DSPE DOPE), Phosph It contains tidylserine (DOPS). Fatty acids include C14:0 and palmitic acid (C1 6:0), Stearic acid (C18:0), Oleic acid (C18:1), Linoleic acid (C1 8:2), linolenic acid (C18:3) and arachidonic acid (C20:4), C20:0, This includes C22:0 and lethicin. In certain embodiments of the present invention In this context, the phospholipids are phosphatidylserine and 1,2-distearoyl-sn-glycerol. -3-Phosphocholine (DSPC), 1,2-Dipalmitreoyl-sn-Glycerol-3- Phosphocholine, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC ), dilauroyl phosphatidylcholine (DLPC), 1,2-dieicosenoyl-sn -Glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine It could be *Drug-Oxygen-Protective Phenomenon* (DOPC).

[0150] In some embodiments, terpine may include, but is not limited to, the following. It is not: monoterpenes, for example geraniol, terpineol, limonene, Myrcene, linalool, or pinene; sesquiterpenes, e.g., humulene, farnesene Farnesol; diterpenes, such as cafestol, carweol, sembrene. and taxadienes, triterpenes, such as squalene and squalante; tetraterpenes For example, acyclic lycopene, monocyclic gamma-carotene, and bicyclic alpha- and Beta-carotenes; polyterpenes and norisoprenoids. In some embodiments, Therefore, terpine is squalene.

[0151] In one embodiment of the present invention, SNE is 50-85 mol% squalene and 1 Contains ~10 mol% of nonionic surfactant. In one embodiment of this embodiment, non The ionic surfactant is a mixture of PS-20 and SPAN-85 or PS-80 and SPAN-85. Contains a mixture with N-85.

[0152] In one embodiment of the present invention, SNE is 0-45 mol% cationic lipid, 50 It contains approximately 85 mol% squalene and 1 to 10 mol% nonionic surfactant. In one embodiment of the embodiment, the nonionic surfactants are PS-20 and SPAN-85 Includes a mixture of or a mixture of PS-80 and SPAN-85.

[0153] General manufacturing methods for SNE (in the presence and absence of cationic lipids) Generally, SNE is produced, for example, by first combining and mixing the lipid components, or It can be formed by first utilizing a single lipid such as a cationic lipid. Mixing Once blended (when mixing lipid components together), add aqueous buffer and the first lipid It is mixed with lipid or viscous components to form a blended emulsion mixture. The prepared emulsion components are first subjected to coarse homogenization, and then The mixture is then subjected to a fine homogenization process. Next, the resulting mixture is subjected to a final filtration process and stored at 4°C. The lipid solution contains one or more cationic lipids, one or more terpenes (e.g., squalene), and one or more Sorbitan-based surfactants (e.g., PS-20 or PS-80; SPAN-8) 5) may be included in a specific molar ratio.

[0154] General manufacturing method of LNP LNPs and methods for producing LNPs are well known in the art. LNPs are publicly known, and the following Listed in the publications: US, 7,691,405, US2006 / 0083780 , US2006 / 0240554, US2008 / 0020058, US2009 / 02 63407, US2009 / 0285881, WO2009 / 086558, WO200 9 / 127060, WO2009 / 132131, WO2010 / 042877, WO2 010 / 054384, WO2010 / 054401, WO2010 / 054405 WO2010 / 054406.

[0155] The method for producing LNP consists of the following four main steps: 1) Lipid mixture and aqueous buff 1) Preparation of solution 2) Split stream mixing 3) NP formation, 4) ultrafiltration, and 5) filtration.

[0156] Generally, lipid components are dissolved in ethanol, and then sterilized and filtered to form a lipid mixture. Several aqueous buffers are also prepared. Then the lipid mixture and the buffer stream are mixed together. Mix using a mixer or Y mixer, then dilute with aqueous buffer immediately after the outlet. This is mixed to form an LNP intermediate. The LNP intermediate is then subjected to ultrafiltration. The substance is concentrated and then diafiltration is performed in an appropriate buffer. Then, remove any remaining ethanol. After diafiltration, reach the final target concentration. To achieve this, a final concentration process is carried out. Next, the LNP bulk is filtered through a sterile filter, and -7 Store frozen at 0°C.

[0157] Pneumococcal conjugate vaccine composition Pneumococcal conjugate vaccines or compositions have already been disclosed. WO2011 / See 100151, WO2019 / 139692 and WO2020 / 131763. I want to.

[0158] Examples of bacterial capsular polysaccharides derived from Streptococcus pneumoniae include serotypes 1 and 2. 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F , 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20(2 0A and 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35 Examples include F or 38.

[0159] General manufacturing methods for capsular polysaccharides A method for producing a pneumococcal conjugate vaccine composition has already been disclosed. WO201 Refer to 1 / 100151, WO2019 / 139692 and WO2020 / 131763. I want to be illuminated.

[0160] Bacterial capsular polysaccharides, especially those used as antigens, are suitable for use in the present invention. Furthermore, they can be easily identified by methods for identifying immunogenic and / or antigenic polysaccharides. Examples of bacterial capsular polysaccharides derived from Streptococcus pneumoniae include serotypes 1 and 2. , 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12 F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20( 20A and 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 3 5F or 38 are examples.

[0161] Polysaccharides can be purified by known techniques. However, this invention relates to polysaccharides purified from natural sources. Polysaccharides are not limited to this method; they can also be obtained by other methods such as total or partial synthesis. Capsular polysaccharides derived from Leptococcus pneumoniae are obtained by standard techniques known to those skilled in the art. It can be manufactured. For example, polysaccharides can be isolated from bacteria and, to some extent, by known methods. For example, see European patent numbers EP497524 and EP497525), preferred Alternatively, microfluidization is achieved using a homogenizer. It can be sized by (tion) or chemical hydrolysis. Streptococcus pneumoniae strain can be grown in soybean-based media. The individual polysaccharides are then purified by standard processes including centrifugation, precipitation, and ultrafiltration. This is possible. For example, U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent See License No. 5,847,112. To reduce viscosity, and / or To improve filterability and lot-to-lot consistency of the subsequent conjugated product, It is possible to size the sugar.

[0162] Using standard techniques, the purified protein is used to introduce functionalities that can react with the carrier protein. It is possible to chemically activate polysaccharides. Chemical activation of polysaccharides and subsequent carriers Conjugation (binding) to proteins is covered by U.S. Patent No. 4,365,170, No. 4. This is achieved by means of those described in Nos. 673,574 and 4,902,506. In short, pneumococcal polysaccharides are treated with periodate-based oxidizing agents, such as periodic acid. Reacting with sodium periodate, potassium periodate, or periodic acid to form adjacent hydroxyls This leads to oxidative cleavage of the group, generating a reactive aldehyde group. Periodate (for example) For example, the appropriate molar equivalent of sodium periodate, sodium metaperiodate, etc. is 0 0.05-0.5 molar equivalents (molar ratio of periodate to polysaccharide repeating units) or 0.1-0. It contains 5 molar equivalents. The reaction of periodate is a reaction of sodium periodate. Diol conformations that control the accessibility of the oxyl group (e.g., acyclic diols, syl Depending on the diol (transdiol), various times ranging from 30 minutes to 24 hours are required. I will.

[0163] The term "periodate" refers to both periodate (periodate) and periodic acid. This term also includes Metaperyodart (IO4 - ) and Orthoperyodart (IO6 - ), including various salts of periodate (e.g., sodium periodate and potassium periodate). The capsular polysaccharide is oxidized in the presence of metaperiodate or in the presence of sodium periodate (NaIO4). Further, the capsular polysaccharide can be oxidized in the presence of orthoperiodate or in the presence of periodic acid.

[0164] The purified polysaccharide can also be linked to a linker. Once each capsular polysaccharide is activated or linked to a linker, it is individually conjugated to a carrier protein to form a glycoconjugate (complex carbohydrate). Polysaccharide conjugates can be prepared by known coupling techniques .

[0165] The polysaccharide is coupled (linked) to a linker, and the free end of the linker is an ester group to form a polysaccharide-linker intermediate. Therefore, the linker is a linker having at least one terminal that is an ester group. The other end is selected such that it can react with the polysaccharide to form a polysaccharide-linker intermediate.

[0166] The polysaccharide can be coupled to the linker using a primary amine group in the polysaccharide. In this case, the linker typically has ester groups at both ends. This allows coupling to occur by reacting one of the ester groups with the primary amine group of the polysaccharide via nucleophilic acyl substitution . This reaction produces a polysaccharide-linker intermediate in which the polysaccharide is coupled to the linker via an amide bond. Therefore, the linker has a first ester group for reacting with the primary amine group of the polysaccharide, and reacts with the primary amine group of the carrier molecule ​​​It is a bifunctional linker that provides a second ester group for esterification. A typical linker is azipi It is N-hydroxysuccinimide diester (SIDEA) of hydroxysuccinimide acid.

[0167] Coupling can also be performed indirectly, that is, coupling to the linker This can be done using an additional linker used to derivatize the polysaccharide before linking.

[0168] Polysaccharides use the carbonyl group at the reducing end of the polysaccharide to couple to an additional linker. This coupling can be performed. This coupling involves the following two steps: (a1) carbonyl (a2) a step of reacting the group with an additional linker, and (a2) the free end of the additional linker with phosphorus The process includes reacting with a ker. In these embodiments, the additional linker is typical It has primary amine groups at both ends, thereby making one of the primary amine groups a polysaccharide. Step (a1) can be carried out by reacting the carbonyl group with reductive amination. To enable this. A primary amine group, which is reactive with the carbonyl group of a polysaccharide, is used. Hydrazide Alternatively, a hydroxylamino group is preferred. The same primary amine group is typically found in polysaccharides. An additional linker is present at both ends, enabling the possibility of Ps)-Ps coupling. The reaction involves the coupling of a polysaccharide to an additional linker via a CN bond. It generates a target linker intermediate.

[0169] Polysaccharides use different groups in the polysaccharide, especially carboxyl groups, to create additional linkers. It can be coupled. This coupling is performed in the following two steps, namely (a1) the base (a2) a step of reacting with an additional linker, and (a2) the free end of the additional linker -Includes a step of reacting with. In this case, the additional linker is typically at both ends. It has a primary amine group, thereby connecting one of the primary amine groups with the carboxyl group of the polysaccharide. Step (a1) can be carried out by initiating the reaction through AC activation. Polysaccharide E A DAC-activated carboxyl group and a reactive primary amine group are used. This is preferable. The same primary amine group is typically present at both ends of the additional linker. The reaction involves the coupling of a polysaccharide to an additional linker via an amide bond. This generates an additional linker intermediate.

[0170] Carrier protein In certain embodiments of the present invention, CRM197 is used as the carrier protein. CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin. 197 is Corynebacterium diphyllum grown in casamino acid and yeast extract-based medium. Phtheria (Corynebacterium diphtheria) strain C7 (b197) It can be isolated from cultures of ). Furthermore, CRM197 is U.S. Patent No. 5,614,382. It can be recombinantly manufactured according to the methods described. Typically, CRM197 is limited Purification by a combination of external filtration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, the CRM197 is a Pfenex Express ion Technology(TM)(Pfenex Inc., San Diego Using CA, Pseudomonas fluorescein (Pseudomonas fluorescein It is manufactured in Pseudomonas fluorescens.

[0171] Other suitable carrier proteins include additional inactivating bacterial toxins, such as DT( Diphtheria toxoid, TT (tetanus toxoid) or TT fragment C, pertussis toxoid Ido, cholera toxoid (for example, International Patent Application Publication No. WO 2004 / 083251) (as listed), Escherichia coli (E. coli) LT, Escherichia coli (E. coli) ST, and Pseudomonas aeruginosa It contains exotoxin A derived from bacterial outer membrane proteins, such as outer membrane protein complex c(O). MPC), porin, transferrin-binding protein, pneumococcus surface protein A(P) spA; see International Patent Application Publication No. WO 02 / 091998), pneumococcus aureus Dohesin protein (PsaA), C5a derived from Group A or Group B Streptococcus. Plunderidase, or Haemophilus influenzae uenzae) Protein D, pneumococcal pneumococcus pneumococcus (Kuo et al., 1995, Infe (ct Immun 63;2706-13), for example, ply detoxified in some way For example, see dPLY-GMBS (International Patent Application Publication No. WO 04 / 081515). (I want to be) or dPLY-formol, PhtX, for example PhtA , fusions of PhtB, PhtD, PhtE and Pht proteins, for example, PhtDE fusion Compounds, PhtBE fusions (International Patent Application Publication Numbers WO 01 / 98334 and WO 0 Other proteins (see 3 / 54007) may also be used. Keyhole lysate, petroleum hemocyanin (KLH), bovine serum albumin (BSA), and This is a purified protein derivative (PPD) of tuberculin, PorB(N. meningiti). (derived from DIS), PD (Haemophilus influenzae protein D; example) For example, see European Patent No. EP 0 594 610 B), or its immunological Functional equivalents, synthetic peptides (European Patent Nos. EP0378881 and EP0427) See 347), heat shock protein (International Patent Application Publication No. WO 93 / 1 See 7712 and WO 94 / 03208), pertussis protein (International Patent See application publication number WO 98 / 58668 and European patent number EP0471177. (Body), cytokines, lymphokines, growth factors or hormones (International Patent Application Publication Number) See WO 91 / 01146), multiple human CDs from antigens of various pathogens. Artificial protein containing 4+ T cell epitopes (Falugi et al., 2001, Eur J See Immunol 31:3816-3824, for example, N19 protein Quality (Baraldoi et al., 2004, Infect Immun 72:4884-7) (See reference), iron uptake protein (International Patent Application Publication No. WO 01 / 72337) (See also) the toxins of Clostridium difficile. A or B (see International Patent Publication No. WO 00 / 61761), and Flake Lynn (Ben-Yedidia et al., 1998, see Immunol Lett 64:9) (The protein that is illuminated can also be used as a carrier protein.)

[0172] When a polyvalent vaccine is used, a secondary carrier is used for one or more of the antigens in the polyvalent vaccine. It can be used. The second carrier protein is preferably non-toxic and non-reactive, and in a sufficient quantity. It is a protein that is available in purity. The second carrier protein also increases the immunogenicity of the antigen. To strengthen it, it is conjugated to an antigen, such as Streptococcus pneumoniae polysaccharide. or conjugate. The carrier protein should conform to standard conjugation methods. Each capsular polysaccharide that is not conjugated to the first carrier protein is the same second carrier. They can be conjugated to body proteins (for example, each capsule polysaccharide molecule can be conjugated to a single carrier protein) (Conjugated to the protein). Not conjugated to the first carrier protein. Capsular polysaccharides can be conjugated to two or more carrier proteins (each capsular polysaccharide molecule is single (It is conjugated to a carrier protein.) In such embodiments, the same blood Each capsule polysaccharide in the clear form is typically conjugated to the same carrier protein. DT mutants, such as the following, can also be used as the second carrier protein: CRM 176, CRM228, CRM45 (Uchida et al., 1973, J Biol Che m 218:3838-3844);CRM9, CRM45, CRM102, CRM10 3 and CRM107, as well as Nicholls and Youle, Genetica lly Engineered Toxins, edited by Frankel, Maecel De Other mutations described in kker Inc, 1992; Glu-14 Deletion of 8 or mutation from there to Asp, Gln or Ser and / or Al a 158 mutation to Gly and U.S. Patent No. 4,709,017 or U.S. Patent No. Those having other mutations disclosed in Patent No. 4,950,740; Lys 516 , at least one of Lys 526, Phe 530 and / or Lys 534 Mutations of residues, as well as U.S. Patent No. 5,917,017 or U.S. Patent No. 6,455 , having other mutations disclosed in Patent No. 6735; or U.S. Patent No. 5,843 Fragments disclosed in No. 711.

[0173] Conjugation by reductive amination The covalent bonding of polysaccharides to carrier proteins is primarily due to the amine-reactive portion on the polysaccharide being the primary component of the protein. This can be carried out by reductive amination, which involves direct bonding to quaternary amine groups (mainly lysine residues). (Well-known) As shown, the reductive amination reaction proceeds in a two-step mechanism. First, the aldehyde of molecule 1... The reaction between the hydride group (R-CHO) and the primary amine group (R'-NH2) of molecule 2 results in the formula A Schiff base intermediate of R-CH=N-R' is formed. In the second step, the Schiff base It is reduced to form an amino compound of formula R-CH2-NH-R'. Numerous reducing agents are available. It can be used, but highly selective reagents such as sodium borohydride (NaCNBH3) The original reagent is the most commonly used because such reagents only contain the imine functional group of Schiff bases. This is because it specifically reduces it.

[0174] All polysaccharides have an aldehyde functional group (terminal aldehyde functional group) at the end of the chain, therefore, Conjugate methods, including reductive amination of sugars, are very commonly applicable and repeatable. If no other aldehyde functional groups (intrachain aldehyde functional groups) exist within the same position, then The method makes it possible to obtain a conjugate in which a polysaccharide molecule is bound to a single molecule of a carrier protein. I'll do that.

[0175] Typical reducing agents are cyanoborohydride salts, such as sodium cyanoborohydride. A commonly used imine-selective reducing agent is sodium borocyanohydride, but Other cyanoboron hydride salts, including potassium anoboron hydride, can also be used. Initial cyanide levels and conjugation reactions in sodium borochemical reagent lots. Differences in residual cyanide levels result in inconsistent conjugation performance. Product characteristics such as conjugate size and conjugate Ps to CRM197 ratio This can cause fluctuations. Controlling the level of free cyanide in the final reaction product and / or By reducing this, the variability of conjugation can be reduced.

[0176] Residual unreacted aldehydes on polysaccharides can be removed, if desired, with a potent agent such as sodium borohydride. It may be reduced by the addition of a reducing agent. Generally, the use of a strong reducing agent is preferred. However, in some polysaccharides, it is preferable to avoid this step. For example, streptococcus S. pneumoniae serotype 5 contains ketone groups that readily react with potent reducing agents. In this case, it is preferable to avoid the reduction step in order to protect the antigenic structure of the polysaccharide.

[0177] After conjugation, concentration / diafiltration, ultrafiltration, precipitation / elution, , one or more of any techniques well known to those skilled in the art, including column chromatography and depth filtration This results in excess conjugation reagents, as well as residual free carrier proteins and free polysaccharides. To remove the polysaccharide-carrier protein conjugate, the polysaccharide-carrier protein conjugate is purified. For example, US patent See Patent No. 6,146,902. In one embodiment, the purification step is ultrafiltration. It is due to the excess.

[0178] Pneumococcal conjugate composition This invention relates to polysaccharide-carrier protein conjugates in the presence or absence of cationic lipids. Pneumonia containing, or essentially consisting of, or comprising SNE The present invention provides a bacterial conjugate composition. The present invention further provides cationic lipids and pharmaceutically acceptable In the presence or absence of the carrier, the polysaccharide-carrier protein conjugate is coupled with SNE. pneumococcal conjugates that contain, essentially consist of, or comprise them The present invention provides compositions. The present invention further provides cationic lipids and, optionally, pharmaceuticals. In the presence or absence of a scientifically acceptable carrier, the polysaccharide-carrier combination described herein Contains any combination of protein conjugates together with SNE, or derived from them The present invention provides pneumococcal conjugate compositions that target or consist of these bacteria. The mature plants are 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, or 35 different polysaccharide-carrier protein conjugates This includes, or is essentially derived from, or consists of, conjugate Each of the components contains a different capsular polysaccharide conjugated to a carrier protein. In one embodiment, the polysaccharide that is part of the polysaccharide-carrier protein conjugate is Serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C of Leptococcus pneumoniae 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16 F, 17F, 18C, 19A, 19F, 20 (20A or 20B), 22F, 23A, This includes, but is not limited to, 23B, 23F, 24F, 33F, 35B, 35F, and 38. It is not that. In another embodiment, the serotype group is 4, 6B, 9V, 14 , including 18C, 19F and 23F, or essentially derived from them, or In another embodiment, the serotype groups are 1, 3, 4, 5, 6A, 6B, and 7. F, 9V, 14, 18C, 19A, 19F and 23F, including or derived from them In another embodiment, the serotype group is 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and It includes, or is essentially derived from, or consists of. Another fruit In terms of application methods, the serotype groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, and 10A. , 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33 F includes, or is essentially made from, or consists of. Another embodiment In this case, the serotype groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F , including 24F, 33F and 35B, or essentially derived from them, or In another embodiment, the serotype groups are 1, 3, 4, 5, 6A, 6B, and 7. F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B, 1 Including 8C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, Or essentially become from them, or consist of them. In another embodiment, The serotype groups are 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, and 12F. , 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, Includes, essentially derived from, or consisting of 33F and 35B. In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N, 10A, 11A , 12F, 15A, 15B, 16F, 17F, 19A, 20A, 22F, 23A, 23B , including 24F, 31, 33F and 35B, or essentially derived from them, or These consist of... In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N , 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19 This includes A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 35B, and It essentially becomes from them, or consists of them. In another embodiment, blood The clean type group consists of 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, and 16. F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 3 5B includes, or essentially consists of, or comprises. Another implementation form In terms of state, the serotype groups are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, and 15. A, 15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, Includes, essentially derived from, or consisting of 33F and 35B. In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N, 10A, 11A , 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20, 22F , including 23A, 23B, 24F, 31, 33F and 35B, or essentially derived from them In another embodiment, the serotype group is 3, 6A , 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A , including 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, or so Essentially consisting of or comprising them. In another embodiment, serotype The group consists of 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B, 24F, 31, 33F and 35B In another embodiment, In this case, the serotype groups are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B , including 24F, 31, 33F and 35B, or essentially derived from them, or These consist of... In another embodiment, the serotype group is 3, 6A, 7F, 8, 9N , 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20B, 22F , including 23A, 23B, 24F, 31, 33F and 35B, or essentially derived from them In another embodiment, the serotype is a carrier protein Quality is conjugated to CRM197. In another embodiment, all The serotype is conjugated to the carrier protein. In another embodiment, The preferred carrier protein is CRM197.

[0179] Instructions for use / Dosage The composition of the present invention, when administered as a vaccine systemically or via a mucosal route, can help prevent infection. For example, it can be used to protect or treat people who are susceptible to pneumococcal infections. In this embodiment, the present invention provides an immunologically effective amount of the composition of the present invention to a human. Immune response to Streptococcus pneumoniae capsular polysaccharide conjugates, including The present invention provides a method for inducing [something]. In another embodiment, the present invention provides a composition of the present invention. A method of administering an immunologically effective dose to a human being for the treatment of pneumococcal infection, including the step of administering an immunologically effective dose to a human being. Provides a method for sowing seeds.

[0180] The optimal amount of each vaccine component is determined by standard procedures including observation of an appropriate immune response in the target population. This can be confirmed by research. For example, in another embodiment, the vaccine is administered to humans. The dosage for each species is determined by extrapolating data from animal studies to human data. Another implementation In this context, the dosage is determined empirically.

[0181] The method of the present invention is for invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections ( Both acute otitis media and sinusitis are caused by Streptococcus pneumoniae. It may be used to prevent and / or alleviate the primary clinical syndromes that may result.

[0182] The composition of the present invention may be administered by injection via intramuscular, intraperitoneal, intradermal, or subcutaneous route, This may include one or more mucosal administration to the oral / gastrointestinal tract, respiratory tract, or urogenital tract. In the embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (because, Nasopharyngeal carriage of Streptococcus pneumoniae can be more effectively prevented, and infection can be mitigated in its early stages. (This is because...)

[0183] The amount of conjugate at each vaccine dose was determined to promote an immune defense response without significant side effects. This can be selected as the amount that induces [a certain condition]. Such an amount may vary depending on the serotype of Streptococcus pneumoniae. Generally, in the case of polysaccharide-based conjugates, each dose is 0.1 to 100 mg, especially 0.1 Each polysaccharide contains approximately 10 mg, more specifically 1-5 mg. For example, each dose is 100, 150 , 200, 250, 300, 400, 500 or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 8, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg It may include.

[0184] In one embodiment, according to any of the methods of the present invention, the subject is a human. In a specific embodiment, human patients include infants (under 1 year old), toddlers (approximately 12-24 months old), and In some embodiments, the patient is a child (approximately 2-5 years old). In other embodiments, the human patient is an elderly patient (65 years old or older). The composition of the present invention is suitable for older children, adolescents and adults (e.g., 18-). It is also suitable for use by those aged 45 or between 18 and 65.

[0185] Therefore, one embodiment of the present invention provides an immunologically effective amount of any of the compositions of the present invention. This includes administering the drug to patients or subjects to control Streptococcus pneumoniae. The invention includes methods for treating or preventing diseases or disorders caused by [the disease]. In this case, the present invention relates to (i) the following (a), (b), (c), (e), (f) or (g (ii) for use in the following (a), (b), (c), (e), (f) and (g) for use as a pharmaceutical or composition for (iii) below a) (b) (c) (e) (f) or (g) manufacture (or production) of pharmaceuticals. For use in the present invention (i.e., the combinations described throughout this specification), Includes: (a) treatment (for example, of the human body), (b) medicine, (c) Streptococcus • Treatment or prevention of infection by Pneumoniae, (e) Streptococcus pneumoniae (f) Prevention of recurrence of infection, pathological symptoms associated with Streptococcus pneumoniae infection Progression, onset or reduction of severity and / or Streptococcus pneumoniae infection The possibility of (g) meningitis, pneumonia, bacteremia, acute otitis media and sinusitis ( Treatment of Streptococcus pneumoniae-related diseases (not limited to these) Treatment, prevention, or delay of its onset, severity, or progression, and Streptococcus nigricans Treatment or prevention of diseases or disorders caused by eumoniers.

[0186] In one embodiment of the method of the present invention, the composition of the present invention is administered as a single dose. In another embodiment, the vaccine is administered in two, three, or at appropriate intervals. It is administered four or more times. For example, the composition is administered at intervals of 1, 2, 3, 4, 5 or 6 months or They can be administered in any combination. The immunization schedule is as follows regarding the pneumococcal vaccine. It is possible to follow the established rules. For example, Streptococcus pneumoniae Standard schedule for infants and toddlers with invasive diseases caused by These are 2 months old, 4 months old, 6 months old, and 12-15 months old. Therefore, preferred embodiment In this case, the composition is divided into four dose series for 2 months, 4 months, 6 months, and 12-15 months of age. It is administered as a drug.

[0187] The composition of the present invention also contains one or more proteins derived from Streptococcus pneumoniae. See. Examples of Streptococcus pneumoniae proteins suitable for inclusion include, In the international patent application publication numbers WO 02 / 083855 and WO 02 / 053761 This includes items that have been identified.

[0188] The formulation of the present invention In some embodiments, SNE and Streptococcus pneumoniae polysaccharide-carrier The present invention provides a composition comprising a protein conjugate. In some embodiments, S NE and at least one, or at least three, or at least seven, or fewer 10 each, or at least 13, or at least 15, or at least 20 1, or at least 24, or at least 27, or at least 30 stocks Streptococcus pneumoniae polysaccharide containing Leptococcus pneumoniae serotype - Provides a composition comprising a carrier protein conjugate. In some embodiments, SNE and 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 streptococci Streptococcus pneumoniae polysaccharide-carrier tannin containing Cass pneumoniae serotype The present invention provides a composition comprising a protein conjugate. In some embodiments, SNE And Streptococcus, consisting of 4, 6B, 9V, 14, 18C, 19F and 23F. Streptococcus pneumoniae polysaccharide-carrier protein containing pneumoniae serotype The present invention provides a composition comprising a conjugate. In some embodiments, the composition comprises SNE and 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F Streptococcus pneumoniae serotypes are found in Streptococcus pneumoniae. The present invention provides a composition comprising a Monier polysaccharide-carrier protein conjugate. Several implementations In terms of state, SNE and 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 1 Streptococcus pneumoniae, consisting of 9A, 19F, 22F, 23F, and 33F. Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing serotype The present invention provides a composition comprising SNE and 1, 3, 4, 5. , 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19 Streptococcus pneumoniae blood consisting of A, 19F, 22F, 23F, and 33F Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing the clear form The present invention provides a composition comprising the following: In some embodiments, SNE and 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C , consisting of 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Streptococcus pneumoniae polysaccharide containing Leptococcus pneumoniae serotype - Provides a composition comprising a carrier protein conjugate. In some embodiments, These are SNE and 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 11A, 12F, 1 4, 15A, de-O-acetyl-15B, 18C, 19A, 19F, 22F, 23B, 23 Includes Streptococcus pneumoniae serotypes F, 24F, 33F, and 35B. Contains a Streptococcus pneumoniae polysaccharide-carrier protein conjugate The composition is provided. In some embodiments, SNE and 1, 3, 4, 5, 6A, 6 B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 2 Streptococcus nyus, consisting of 2F, 23B, 23F, 24F, 33F and 35B Streptococcus pneumoniae polysaccharide-carrier protein containing Monier serotype The present invention provides a composition comprising jugate. In some embodiments, SNE and 1, 3 , 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, Streets consisting of 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Streptococcus pneumoniae polysaccharide containing the serotype of Ptococcus pneumoniae - The present invention provides a composition comprising a carrier protein conjugate. In some embodiments, SNE and 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A De-O-acetyl-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F , containing Streptococcus pneumoniae serotypes consisting of 33F and 35B A composition containing a Leptococcus pneumoniae polysaccharide-carrier protein conjugate is proposed. Provided. In some embodiments, SNE and 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23 Streptococcus pneumoniae blood consists of B, 23F, 24F, 33F, and 35B Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing the clear form The present invention provides a composition comprising the following: In some embodiments, SNE and 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C , consisting of 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Streptococcus pneumoniae polysaccharide containing Leptococcus pneumoniae serotype - Provides a composition comprising a carrier protein conjugate. In some embodiments, These are SNE and 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, de-O-acetyl-15B, 18C, 19A, 19F, 22F, 23B, Streptococcus pneumoniae serotypes consisting of 23F, 24F, 33F, and 35B Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing The present invention provides compositions containing SNE and 1, 3, 4, 5, 6A. , 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 1 Strep consisting of 9A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Streptococcus pneumoniae polysaccharide containing Tococcus pneumoniae serotype The present invention provides a composition comprising a somatic protein conjugate. In some embodiments, SNE and 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16 F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F and 3 Streptococcus pneumoniae serotype 5B is included in Streptococcus pneumoniae. The present invention provides compositions comprising a Pneumoniae polysaccharide-carrier protein conjugate. In this embodiment, SNE and 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20A, 22F, 23A Streptococcus pneumoniae, consisting of 23B, 24F, 31, 33F and 35B Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing the E serotype The present invention provides a composition containing SNE and 3, 6A, 7. F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 2 Strepto consists of 0A, 22F, 23A, 23B, 24F, 31, 33F and 35B. Streptococcus pneumoniae polysaccharide carrier containing Coccus pneumoniae serotype The present invention provides a composition comprising a protein conjugate. In some embodiments, S NE, 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F , 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35B Streptococcus pneumoniae serotype, which consists of Streptococcus pneumoniae The present invention provides a composition comprising a Monier polysaccharide-carrier protein conjugate. Several implementations In terms of form, SNE, 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15 A, de-O-acetylation - 15B, 16F, 17F, 19A, 20, 22F, 23A, 23 Streptococcus pneumoniae serum consisting of B, 24F, 31, 33F, and 35B Streptococcus pneumoniae polysaccharide-carrier protein conjugate containing the type The present invention provides a composition containing SNE and 3, 6A, 7F, 8 , 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 2 Streptococcus consisting of 2F, 23A, 23B, 24F, 31, 33F, and 35B • Streptococcus pneumoniae polysaccharide-carrier protein containing the pneumoniae serotype The present invention provides a composition comprising a quality conjugate. In some embodiments, SNE and 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F , from 19A, 20B, 22F, 23A, 23B, 24F, 31, 33F and 35B Streptococcus pneumoniae containing serotype The present invention provides a composition comprising a polysaccharide-carrier protein conjugate. In several embodiments, In this regard, SNE and 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, O-acetylation - 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B, The Streptococcus pneumoniae serotypes consist of 24F, 31, 33F, and 35B. Contains Streptococcus pneumoniae polysaccharide-carrier protein conjugate The present invention provides a composition containing SNE and 3, 6A, 7F, 8, 9 N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20B, 22 Streptococcus species consisting of F, 23A, 23B, 24F, 31, 33F, and 35B. Streptococcus pneumoniae polysaccharide-carrier protein containing pneumoniae serotype The present invention provides a composition containing a conjugate.

[0189] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. This invention provides a pneumococcal conjugate composition containing a Streptococcus pneumoniae and a stable nanoemulsion (SNE). ru.

[0190] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. We propose a pneumococcal conjugate composition containing Streptococcus pneumoniae, SNE, and a pharmaceutically acceptable carrier. To provide.

[0191] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. To, emulsifiers and / or solubilizers and / or surfactants and, if desired, SNE containing cationic lipids or mixtures thereof, and pneumococcal conjugate A gate composition is provided.

[0192] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. and surfactants and / or terpenes and / or cationic lipids or their The present invention provides a pneumococcal conjugate composition comprising a mixture of SNEs.

[0193] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. T, and one or more surfactants and one or more terpenes and one which may be used as desired. The present invention provides a pneumococcal conjugate composition comprising a cationic lipid-containing SNE.

[0194] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. It may be used with 1 to 3 surfactants and 1 to 3 terpenes as desired. The present invention provides a pneumococcal conjugate composition containing a SNE containing 1 to 3 cationic lipids. do.

[0195] This invention further relates to Streptococcus pneumoniae polysaccharide-carrier protein conjugates. It may also contain 1-2 surfactants and 1-2 terpenes, and may be used as desired. The present invention provides a pneumococcal conjugate composition comprising a SNE containing 1-2 cationic lipids. do.

[0196] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Lungs, and optionally, iv) lungs containing cationic lipids and SNEs We provide a Streptococcus conjugate composition.

[0197] This invention relates to 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate (i) Sorbitan triolet (SPAN-85), (ii) Polysorbate -20 (PS-20), iii) squalene, and iv) which may be used as desired. The present invention provides a pneumococcal conjugate composition comprising a cationic lipid-containing SNE.

[0198] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squam Pneumonia comprising SNE containing a cationic lipid, which may be used optionally. The present invention provides a coccus conjugate composition in which the cationic lipid is lipid nanoparticle (LNP). I haven't met with them.

[0199] The present invention further includes 1) Streptococcus pneumoniae polysaccharide-carrier protein conjugate Gate and 2)i) Sorbitan Triolet Art (SPAN-85), (ii) Polysorbate iii) Squalene and iv) May be used as desired. We provide a pneumococcal conjugate composition comprising a cationic lipid-containing SNE, and here Cationic lipids do not associate with lipid nanoparticles (LNPs).

[0200] The present invention further relates to the above-mentioned Streptococcus pneumoniae conjugate composition comprising a pharmaceutically acceptable carrier. provide.

[0201] In some embodiments, the composition is such that the carrier protein is CRM197. Contains a polysaccharide-carrier protein conjugate.

[0202] In one embodiment of the composition, SNE comprises PS-20. Another embodiment In terms of form factor, SNE includes the PS-80.

[0203] In one embodiment, the composition comprises a cationic lipid CLA, CLX, or CLY. nothing.

[0204] In one embodiment, the composition comprises a cationic lipid CLA.

[0205] In one embodiment, the composition includes DLinDMA, DLinKC2DMA, and DL Selected from in-MC3-DMA, CLinDMA, and S-octylCLinDMA It contains cationic lipids.

[0206] In one embodiment, Streptococcus pneumoniae polysaccharide-carrier protein The conjugate is a conjugate of the carrier protein CRM197, 1, 2 , 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12 F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20( 20A and 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 3 One of (but not limited to) serotypes selected from 5F and 38 It contains polysaccharides of a specific Streptococcus pneumoniae serotype. Another implementation Morphologically, Streptococcus pneumoniae polysaccharide-carrier protein conjugate Serotypes 4 and 6B are all conjugated to the carrier protein CRM197. , including 9V, 14, 18C, 19F and 23F, or essentially derived from them, or consists of them. In another embodiment, the serotype is the carrier protein CRM Serotypes 1, 3, 4, 5, 6A, 6B, and 7F, all of which are conjugated in 197, Including 9V, 14, 18C, 19A, 19F, and 23F, or essentially derived from them. or consisting thereof. In another embodiment, the serotype is the carrier protein Serotypes 1, 3, 4, 5, 6A, and 6B are all conjugated in CRM197. This includes 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, as well as It essentially becomes from them, or consists of them. In another embodiment, blood The clear form consists of serotypes 1 and 3, in which all cells are conjugated to the carrier protein CRM197. , 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18 Including C, 19A, 19F, 22F, 23F, and 33F, or essentially derived from them or consisting thereof. In another embodiment, the serotype is the carrier protein Serotypes 1, 3, 4, 5, 6A, and 6B are all conjugated in CRM197. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 1 Including or derived from 9F, 22F, 23B, 23F, 24F, 33F and 35B Essentially, or consisting of them. In another embodiment, the serotype is the carrier. Serotypes 1, 3, 4, 5, and 6 are all conjugated to the protein CRM197. A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-oacetylation -15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 3 5B includes, or essentially consists of, or comprises. Another implementation form In this state, all serotypes are conjugated to the carrier protein CRM197. Present serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 1 4, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33 Includes F and 35B, or essentially consists of them. In one embodiment, the serotype is conjugated to the carrier protein CRM197. Serotypes that have been sterilized are 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 1 5B, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33 Includes F and 35B, or essentially consists of them. In one embodiment, the serotype is conjugated to the carrier protein CRM197. Serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, and 15A are deactivated. O-acetylation - 15B, 16F, 17F, 19A, 20A, 22F, 23A, 23B, Including 24F, 31, 33F, and 35B, or essentially derived from them, or It consists of the following. In another embodiment, the serotype is the carrier protein CRM197 All serotypes 3, 6A, 7F, 8, 9N, 10A, and 11A are conjugated. 12F, 15A, 15C, 16F, 17F, 19A, 20A, 22F, 23A, 23B, Including 24F, 31, 33F, and 35B, or essentially derived from them, or It consists of the following. In another embodiment, the serotype is the carrier protein CRM197 All serotypes 3, 6A, 7F, 8, 9N, 10A, and 11A are conjugated. 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 2 Including 4F, 31, 33F and 35B, or essentially derived from them, or those It consists of. In another embodiment, the serotype is fully connected to the carrier protein CRM197. Serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 1 are conjugated. 2F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20, 22F, 2 Including 3A, 23B, 24F, 31, 33F, and 35B, or essentially derived from them or consisting thereof. In another embodiment, the serotype is the carrier protein Serotypes 3, 6A, 7F, 8, 9N, and 1 are all conjugated into CRM197. 0A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 23 A, 23B, 24F, 31, 33F, and 35B, or essentially derived from them. , or consisting thereof. In another embodiment, the serotype is carrier protein C Serotypes 3, 6A, 7F, 8, 9N, and 10 are all conjugated into RM197. A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20B, 22F, 23 A, 23B, 24F, 31, 33F, and 35B, or essentially derived from them. , or consisting thereof. In another embodiment, the serotype is carrier protein C Serotypes 3, 6A, 7F, 8, 9N, and 10 are all conjugated into RM197. A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 2 Including 0B, 22F, 23A, 23B, 24F, 31, 33F and 35B, or Essentially consisting of or comprising. In another embodiment, the serotype is Serotypes 3, 6A, and 7 are all conjugated to the carrier protein CRM197. F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 2 Including 0B, 22F, 23A, 23B, 24F, 31, 33F and 35B, or To essentially become from, or consist of, them.

[0207] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated, and the composition is sorbitan trioleate (SPAN-85), polysorbate Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), and Suku This also includes Allen.

[0208] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further includes TO-80 (PS-80) and squalene.

[0209] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) The further comprises polysorbate-80 (PS-80) and squalene.

[0210] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), and It also contains squalene.

[0211] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains Bate-80 (PS-80) and squalene.

[0212] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), and further comprising squalene.

[0213] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains Bate-80 (PS-80) and squalene.

[0214] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), and further containing squalene.

[0215] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po It further contains resorbate-80 (PS-80) and squalene.

[0216] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), and further containing squalene.

[0217] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated, and the composition is sorbitan trioleate (SPAN-85), polysorbate Rubate-20 (PS-20) or Polysorbate-80 (PS-80), Squalene , and further comprising cationic lipids.

[0218] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further contains To-80 (PS-80), squalene, and cationic lipids.

[0219] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) It further contains polysorbate-80 (PS-80), squalene, and cationic lipids. nothing.

[0220] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), It further contains allenes and cationic lipids.

[0221] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipids.

[0222] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), squalene, and cationic lipids It also includes.

[0223] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipids.

[0224] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipids are further included.

[0225] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po It further contains resorbate-80 (PS-80), squalene, and cationic lipids.

[0226] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipids are further included.

[0227] In one embodiment, the present invention provides seven different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing an ethyl polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharides are found in serotypes 4, 6B, 9V, 14, and 18C. It consists of 19F and 23F, and all serotypes are conjugated to the carrier protein CRM197. It is gated, and the composition is sorbitan trioleate (SPAN-85), polysorbate Rubate-20 (PS-20) or Polysorbate-80 (PS-80), Squalene , and further comprising the cationic lipid CLA.

[0228] In one embodiment, the present invention provides 13 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 9V, 14, 18C, 19A, 19F and 23F are all serotypes. It is conjugated to the body protein CRM197, and the composition is sorbitan trio Laato (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate It further contains To-80 (PS-80), squalene, and cationic lipid CLA.

[0229] In one embodiment, the present invention provides 15 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. It consists of 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, All serotypes are conjugated to the carrier protein CRM197, and the composition Sorbitan Triolet (SPAN-85), Polysorbate-20 (PS-20) Alternatively, polysorbate-80 (PS-80), squalene, and cationic lipid CLA It also includes.

[0230] In one embodiment, the present invention provides 20 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, It consists of 22F, 23F, and 33F, and all serotypes are linked to the carrier protein CRM197. It is denjugate-treated, and the composition is sorbitan trioleate (SPAN-85), Polysorbate-20 (PS-20) or Polysorbate-80 (PS-80), The material further contains allene and the cationic lipid CLA.

[0231] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipid CLA.

[0232] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-acetylation - 15B From 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B Therefore, all serotypes are conjugated to the carrier protein CRM197, and the set The finished products are sorbitan trioleate (SPAN-85) and polysorbate-20 (PS-2 0) or polysorbate-80 (PS-80), squalene, and cationic lipid C This also includes LA.

[0233] In one embodiment, the present invention provides 24 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 1, 3, 4, 5, 6A, and 6B. , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, It consists of 19F, 22F, 23B, 23F, 24F, 33F, and 35B, and all serotypes. It is conjugated to the carrier protein CRM197, and the composition is sorbitan Rioleart (SPAN-85), Polysorbate-20 (PS-20), or Polysol It further contains β-80 (PS-80), squalene, and cationic lipid CLA.

[0234] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipid CLA further comprise.

[0235] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, De-O-acetylation - 15B, 16F, 17F, 19A It consists of 20, 22F, 23A, 23B, 24F, 31, 33F and 35B, and all The serotype is conjugated to the carrier protein CRM197, and the composition is sorbitan. Tantriolet (SPAN-85), Polysorbate-20 (PS-20) or Po The following further contains resorbate-80 (PS-80), squalene, and cationic lipid CLA. nothing.

[0236] In one embodiment, the present invention provides 21 different Streptococcus pneumoniae. We provide a pneumococcus conjugate composition containing a Nie polysaccharide-carrier protein conjugate. Here, Streptococcus pneumoniae polysaccharide is found in serotypes 3, 6A, 7F, 8, and 9N. 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 2 It consists of 3A, 23B, 24F, 31, 33F and 35B, and all serotypes are carrier proteins It is conjugated with sorbitan trioleate, and the composition is sorbitan trioleate. (SPAN-85), Polysorbate-20 (PS-20), or Polysorbate-80 (PS-80), squalene, and cationic lipid CLA further comprise.

[0237] In one embodiment of the composition, SNE comprises PS-20. Another embodiment In terms of form factor, SNE includes the PS-80.

[0238] In some embodiments, cationic lipids are present in concentrations ranging from approximately 2 μg / mL to approximately 400 mg / mL. and at least one Streptococcus pneumoniae polysaccharide-carrier protein condyl The present invention provides a composition containing conjugates, where each conjugate is approximately 0.02 μg / It exists at a concentration of approximately 200 μg / mL.

[0239] In some embodiments, cationic concentrations range from approximately 0.04 μg / mL to approximately 80 mg / mL. Lipids and at least one Streptococcus pneumoniae polysaccharide-carrier protein The present invention provides a composition comprising a conjugate, wherein each of the conjugates is approximately 0.004 It exists at concentrations ranging from μg / mL to approximately 40 μg / mL.

[0240] In some embodiments, cationic concentrations range from approximately 100 μg / mL to approximately 4.2 mg / mL. Lipids and at least one Streptococcus pneumoniae polysaccharide-carrier protein The present invention provides a composition comprising a conjugate, wherein each of the conjugates is approximately 0.004 It exists at concentrations ranging from μg / mL to approximately 40 μg / mL.

[0241] In some embodiments, the cationic lipid concentration is approximately 100 μg / mL to approximately 20 mg / mL. Quality and at least one Streptococcus pneumoniae polysaccharide-carrier protein We provide a composition containing a conjugate, where each conjugate is approximately 0.004 μm It exists at concentrations ranging from g / mL to approximately 40 μg / mL.

[0242] In some embodiments, cationic lipids are present in concentrations ranging from approximately 2 μg / mL to approximately 400 mg / mL. and at least one Streptococcus pneumoniae polysaccharide-carrier protein condyl A composition comprising a conjugate is provided, wherein each of the conjugates is co-freezed (c Prepared as an o-lyophilized formulation, approximately 0.02 μg / mL to approximately 200 μg It exists at a concentration of g / mL.

[0243] In some embodiments, cationic lipids are present in concentrations ranging from approximately 2 μg / mL to approximately 400 mg / mL. , aluminum in the form of APA at 2 μg / mL to approximately 2 mg / mL and at least one A composition containing a Streptococcus pneumoniae polysaccharide-carrier protein conjugate Provided, here, each of the conjugates is prepared as a co-freezymized formulation of approximately 0. It exists at concentrations ranging from 0.2 μg / mL to approximately 200 μg / mL.

[0244] In some embodiments, the cationic lipid concentration is approximately 20 μg / mL to 2.4 mg / mL. Quality and at least one Streptococcus pneumoniae polysaccharide-carrier protein The present invention provides a composition containing jugate, where each conjugate is approximately 0.2 μg / It exists at a concentration of approximately 24 μg / mL.

[0245] In some embodiments, the cationic lipid concentration is approximately 60 μg / mL to approximately 2.4 mg / mL. Quality and at least one Streptococcus pneumoniae polysaccharide-carrier protein The present invention provides a composition containing jugate, where each conjugate is approximately 0.2 μg / It exists at a concentration of approximately 24 μg / mL.

[0246] In some embodiments, as emphasized in the various embodiments described above, It contains cationic lipids ranging from approximately 0.1 μg / mL to approximately 400 mg / mL, and furthermore, SPAN-85 The present invention provides a composition comprising PS-20 or PS-80 and squalene. In the application form, the cationic lipid is CLA. In another embodiment, The thionic lipid is CLX. In another embodiment, the cationic lipid is CLY That is the case.

[0247] In some embodiments, as emphasized in the various embodiments described above, It contains approximately 60 μg / mL to 2.4 mg / mL of cationic lipids, and further 6 μg / mL to 2 SPAN-85 at 40 μg / mL, PS-20 or PS at 6 g / mL to 240 g / mL The present invention provides compositions containing squalene at -80 and 60 g / mL to 2.4 mg / mL. In another embodiment, the cationic lipid is CLA. In this embodiment, the cationic lipid is CLX. In another embodiment, cationic Lipids are CLY.

[0248] In some embodiments, SPAN-85, 6 μg / mL to 24 mg / mL PS-20 or PS-80 with a concentration of μg / mL to 24 mg / mL, and 60 μg / mL to 2 40 mg / mL squalene and at least one Streptococcus pneumoniae We provide a composition comprising a polysaccharide-carrier protein conjugate, wherein the conjugate Each of these is present at concentrations ranging from approximately 0.02 μg / mL to approximately 200 μg / mL.

[0249] In some embodiments, SPAN-85, 6 μg / mL to 24 mg / mL PS-20 or PS-80 with a concentration of μg / mL to 24 mg / mL, and 60 μg / mL to 2 40 mg / mL squalene and at least one Streptococcus pneumoniae We propose a composition comprising a polysaccharide-carrier protein conjugate, wherein the conjugate Each exists at concentrations ranging from approximately 0.004 μg / mL to approximately 40 μg / mL.

[0250] In some embodiments, SPAN-85, 6 μg / mL to 24 mg / mL PS-20 or PS-80 with a concentration of μg / mL to 24 mg / mL, and 60 μg / mL to 2 40 mg / mL squalene and at least one Streptococcus pneumoniae We provide a composition comprising a polysaccharide-carrier protein conjugate, wherein the conjugate Each of these is present at concentrations ranging from approximately 0.004 μg / mL to approximately 40 μg / mL.

[0251] In some embodiments, SPAN-85 is used in concentrations of approximately 2 μg / mL to 24 mg / mL. PS-20 or PS-80 at μg / mL to 2.4 mg / mL, and 20 μg / mL to 24 mg / mL squalene and at least one Streptococcus pneumoniae We provide a composition comprising a polysaccharide-carrier protein conjugate, wherein the conjugate Each of these is present at concentrations ranging from approximately 0.004 μg / mL to approximately 40 μg / mL.

[0252] In some embodiments, SPAN-85 is present in concentrations of approximately 6 μg / mL to 2.4 mg / mL. PS-20 or PS-80 at 6 μg / mL to 2.4 mg / mL, and 60 μg / mL ~24 mg / mL of squalene, and at least one Streptococcus pneumoniae We provide a composition comprising a Nie polysaccharide-carrier protein conjugate, wherein the conjugate Each of these is prepared as a co-freeze-dried formulation with a concentration of approximately 0.02 μg / mL to 200 μg. It exists at a concentration of / mL.

[0253] In some embodiments, the cationic lipid concentration is approximately 20 μg / mL to 2.4 mg / mL. Quality and at least one Streptococcus pneumoniae polysaccharide-carrier protein The present invention provides a composition containing jugate, where each conjugate is approximately 0.2 μg / It exists at a concentration of approximately 24 μg / mL.

[0254] In some embodiments, SPAN-85, 6 μg / mL to 2.4 mg / mL PS-20 or PS-80 at μg to 2.4 mg / mL, and 60 μg to 24 mg / mL. g / mL squalene and at least one Streptococcus pneumoniae polysaccharide - Provides a composition comprising a carrier protein conjugate, wherein the conjugate is These substances exist at concentrations ranging from approximately 0.2 μg / mL to approximately 24 μg / mL.

[0255] In some embodiments, SPAN-85 is used in concentrations of 6 μg / mL to 2.4 mg / mL. PS-20 or PS-80 at μg / mL to 2.4 mg / mL, and 60 μg / mL to 24 mg / mL squalene and at least one Streptococcus pneumoniae We provide a composition comprising a polysaccharide-carrier protein conjugate, wherein the conjugate Each of these is present at concentrations ranging from approximately 0.2 μg / mL to approximately 24 μg / mL.

[0256] In some embodiments, as emphasized in the various embodiments described above, The composition consists of SPAN-85 at 6 μg / mL to 24 mg / mL and P at 6 μg to 24 mg / mL. Contains S-20 or PS-80, and squalene in a concentration of 60 μg / mL to 240 mg / mL. nothing.

[0257] In some embodiments, as emphasized in the various embodiments described above, The composition includes SPAN-85 at 6 μg / mL to 2.4 mg / mL, and 6 μg / mL to 2.4 mg PS-20 or PS-80 at 60 μg / mL and squamous acid at 60 μg / mL to 24 mg / mL Includes Len.

[0258] In some embodiments, as emphasized in the various embodiments described above, It contains approximately 30 μg / mL to 2.4 mg / mL of cationic lipids, and further 6 μg / mL to 1 SPAN-85 at 4 mg / mL, PS-20 or PS-8 at 6 g / mL to 14 mg / mL The present invention provides compositions containing 0 and squalene in amounts ranging from 60 g / mL to 34 mg / mL. In one embodiment, the cationic lipid is CLA. In another embodiment... In one embodiment, the cationic lipid is CLX. In another embodiment, the cationic lipid is It is CLY.

[0259] In some embodiments, as emphasized in the various embodiments described above, It contains approximately 60 μg / mL to 2.4 mg / mL of cationic lipids, and further 6 μg / mL to 1 SPAN-85 at 4 mg / mL, PS-20 or PS- at 6 g / mL to 14 mg / mL The present invention provides compositions containing 80 and 60 g / mL to 34 mg / mL of squalene. In one embodiment, the cationic lipid is CLA. In another embodiment, In this embodiment, the cationic lipid is CLX. In another embodiment, the cationic lipid It is CLY.

[0260] In some embodiments, SPAN-85 is used in concentrations of 6 μg / mL to 14 mg / mL, and 6 μg / mL is used. PS-20 or PS-80 with a concentration of g / mL to 14 mg / mL, and 60 μg / mL to 34 mg / mL squalene and at least one Streptococcus pneumoniae polyphosphate The present invention provides a composition comprising a sugar-carrier protein conjugate, wherein the conjugate Each exists at a concentration of approximately 0.2 μg / mL to approximately 24 μg / mL.

[0261] The composition of the present invention may be administered subcutaneously, topically, orally, on the mucous membrane, intravenously, or intramuscularly. The composition is administered in an amount sufficient to induce a defensive response. The composition is administered through various routes. For example, it may be administered orally, parenterally, subcutaneously, intramucosally, or intramuscularly. The dose administered is This may vary depending on the patient's overall condition, sex, weight, age, and route of administration.

[0262] The compositions of the present invention, as highlighted in the various embodiments described above, are immunogenic compositions. It can be called a finished product.

[0263] The compositions of the present invention, as highlighted in the various embodiments described above, are vaccines It may also be called a vaccine composition.

[0264] In one embodiment, SNE uses PS-20, sorbitan trioleate, and squamous acid. Len and (13Z,16Z)-N,N-dimethyl-3-nonyldocosa 13,16-di A composition containing an en-1-amine is provided.

[0265] In one embodiment, SNE is 5-15 mol% sorbitan trioleate, 2 5-35 mol% PS-20 or PS-80, 1-2.5 mol% squalene and 5 5-65 mol% of (13Z,16Z)-N,N-dimethyl-3-nonyldocosa 13,1 A composition containing 6-diene-1-amine is provided.

[0266] In one embodiment, the SNE containing cationic lipids is cationic up to 75 mol% Lipids, up to 30 mol% sorbitan trioleate, up to 30 mol% polysorbate A combination containing polysorbate -20 or polysorbate -80 and squalene in amounts of 25-85 mol%, To provide finished products.

[0267] In one embodiment, the SNE containing cationic lipids is cationic up to 50 mol% Lipids, up to 10 mol% sorbitan trioleate, up to 10 mol% polysorbate -20 or polysorbate-80 and a combination containing squalene in amounts of 50-80 mol%, To provide finished products.

[0268] In one embodiment, the SNE containing cationic lipids is cationic up to 24 mol% Lipids, 1-8 mol% sorbitan trioleate, 1-8 mol% polysorbate-2 A composition containing 0 or polysorbate-80 and 60-75 mol% of squalene. To provide.

[0269] In one embodiment, the SNE containing cationic lipids is present in a cationic lipid concentration of approximately 10-14 mol%. Onopropyl lipids, 1-4 mol% sorbitan trioleate, 1-4 mol% polysorbate -20 or polysorbate-80 and a combination containing squalene in amounts of 50-80 mol%, To provide finished products.

[0270] In one embodiment, the cationic lipid-containing SNE is 30-65 mol% Non-stick lipids, 5-30 mol% sorbitan trioleate, 10-40 mol% squalene The present invention provides a composition comprising 0.5 to 4 mol% of PS-20 or PS-80.

[0271] In one embodiment, the cationic lipid-containing SNE is 55-65 mol% Non-stick lipids, 5-15 mol% sorbitan trioleate, 25-35 mol% squalene The present invention provides a composition comprising 1 to 2.5 mol% of PS-20 or PS-80.

[0272] In one embodiment, the cationic lipid-containing SNE is 13-45 mol% Non-stick lipids, 2-4 mol% sorbitan trioleate, 50-82 mol% squalene The present invention provides a composition containing 1.5 to 3 mol% of PS-20 or PS-80.

[0273] In one embodiment, the cationic lipid-containing SNE is 13-14 mol% Non-stick lipids, 1-2 mol% sorbitan trioleate, 79-81 mol% squalene The present invention provides a composition containing 1 to 2 mol% of PS-20 or PS-80.

[0274] In one embodiment, a cationic lipid containing a cationic lipid with 0 mol% SNE is used. , 8-10 mol% sorbitan trioleate, 80-84 mol% squalene and 8 The present invention provides a composition containing approximately 10 mol% of PS-20 or PS-80.

[0275] In one embodiment, the cationic lipid-containing SNE is 20 mol% cationic lipid Quality, 30 mol% sorbitan trioleate, 20 mol% squalene and 30 mol% The present invention provides a composition comprising PS-20 or PS-80.

[0276] In one embodiment, the SNE containing cationic lipids is approximately 2 mol% cationic lipid. Quality, approximately 8 mol% sorbitan trioleate, approximately 82 mol% squalene and approximately 8 mol The present invention provides a composition containing % PS-20 or PS-80.

[0277] In one embodiment, the cationic lipid contains 2 mol% of SNE. , 8 mol% sorbitan trioleate, 82 mol% squalene and 8 mol% PS The present invention provides a composition comprising -20 or PS-80.

[0278] In one embodiment, the SNE containing cationic lipids is approximately 13.82 mol% Occlusive lipids, approximately 3.43 mol% sorbitan trioleate, approximately 80.07 mol% squalane The present invention provides a composition comprising allene and approximately 2.68 mol% of PS-20 or PS-80. .

[0279] In one embodiment, the cationic lipid-containing SNE is 13.82 mol% Non-stick lipids, 3.43 mol% sorbitan trioleate, 80.07 mol% squalene The present invention provides a composition comprising 2.68 mol% of PS-20 or PS-80.

[0280] In one embodiment, the cationic lipid-containing SNE is approximately 44.5 mol% Non-stick lipids, approximately 2.21 mol% sorbitan trioleate, approximately 51.56 mol% squalane The present invention provides a composition comprising len and approximately 1.72 mol% of PS-20 or PS-80.

[0281] In one embodiment, the SNE containing cationic lipids is 44.5 mol% cation Lipids, 2.21 mol% sorbitan trioleate, 51.56 mol% squalene The present invention provides a composition comprising 1.72 mol% of PS-20 or PS-80.

[0282] In one embodiment, SNE is 32 mol% squalene and SPAN is 34 mol% The present invention provides a composition comprising -85 and 34 mol% of PS-20 or PS-80.

[0283] In one embodiment, SNE is 98 mol% squalene, 1 mol% SPAN- The present invention provides a composition containing 85% and 1 mol% of PS-20 or PS-80.

[0284] In one embodiment, SNE is 86 mol% squalene, 7 mol% SPAN- The present invention provides compositions containing 85% and 7 mol% of PS-20 or PS-80.

[0285] In one embodiment, SNE is 92 mol% squalene, 4 mol% SPAN- The present invention provides a composition containing 85% and 4 mol% of PS-20 or PS-80.

[0286] In one embodiment, SNE is 94 mol% squalene, 3 mol% SPAN- The present invention provides a composition containing 85% and 3 mol% of PS-20 or PS-80.

[0287] In one embodiment, SNE is 92.91 mol% squalene, 3.98 mol% A composition comprising SPAN-85 and 3.11 mol% of PS-20 or PS-80 provide.

[0288] In one embodiment, SNE is 62 mol% squalene and SPAN is 17 mol% The present invention provides a composition comprising -85% and 17 mol% of PS-20 or PS-80.

[0289] In each of the above embodiments, the composition is Streptococcus pneumoniae polysaccharide. - Further contains a carrier protein conjugate.

[0290] The present invention provides a method for treating or preventing pneumococcal disease by administering the aforementioned composition.

[0291] The present invention provides the use of the composition for treating or preventing pneumococcal disease.

[0292] All publications cited herein refer to the methods and that may be used in connection with the present invention. For the purpose of describing and disclosing the materials, they are incorporated herein by reference. Nothing in this regard means that the present invention has the right to precede such disclosure due to prior art. This should not be interpreted as an admission that it does not exist.

[0293] Various embodiments of the present invention are described herein with reference to the attached drawings, but the present invention This invention is not limited to those exact embodiments, but is defined in the attached claims. Various modifications and modifications can be made by those skilled in the art without departing from the scope or spirit of the clarity. It should be understood as [this].

[0294] The following examples illustrate the present invention and are not intended to limit it.

[0295] Examples Example 1: Conjugation of Streptococcus pneumoniae polysaccharide-carrier protein using DMSO conjugation Gate manufacturing Dissolve the polysaccharides (those highlighted in the following section, table, and examples) and the target molecule The solution was sized to the required volume, chemically activated, and buffered by ultrafiltration. The sugar and purified CRM197 were separately freeze-dried and redissolved in DMSO. Then, they were redissolved. The polysaccharide solution and CRM197 solution were combined and conjugated as described below. The resulting conjugate is subjected to ultrafiltration and subsequent final 0.2 micron filtration. Therefore, it was purified. In order to produce a conjugate with desirable properties, each step was performed Several process parameters, such as pH, temperature, concentration, and time, were controlled.

[0296] Polysaccharide size reduction Purified pneumococcal capsular polysaccharide (also known as "Ps") powder was dissolved in water. Excluding ST-19A (serotype also referred to as "ST"), which was not reduced, the dissolved polysaccharides were reduced to 0. Filtered through a 45 micron filter, then subjected to either homogenization or acid hydrolysis. The molecular weight of Ps was reduced by adding it. Homogenization was achieved by controlling the pressure and number of passes. The target Ps size was achieved. By controlling the temperature and time, acid hydrolysis The target Ps size was achieved. The polysaccharide was then filtered through 0.2 microns, concentrated, and 5 or Using a 10kDa NMWCO tangential flow ultrafiltration membrane, diafiltration treatment is performed on water. I did.

[0297] De-o-acetylation The size-reduced ST-15B Ps solution was heated to 60°C and then bubbled with sodium bicarbonate buffer ( (pH 9.4) was added to a final concentration of 50 mM. The batch was incubated at 60°C. -The acetyl group was liberated. Potassium phosphate buffer (pH 6) was added to neutralize the pH. The solution was then cooled to ambient temperature. The solution was then concentrated and a 5 or 10 kDa NMWCO solution was added. Diafiltration was performed on the water using a tangential flow ultrafiltration membrane.

[0298] Deketalization (ST-4 only) Size-reduced ST-4 Ps solution in sodium acetate buffer at 50°C and pH 4.1 The polysaccharide was partially deketalized by adjusting the solution. Then, the polysaccharide solution was cooled to 22°C, The activation process was initiated.

[0299] Oxidation of polysaccharides Prepare the polysaccharide solution at 22°C for all serotypes except ST-5, 7F, and 19F. ST-5, 7F, and 19F were adjusted to 4°C. Also, the size reduction of polysaccharides due to activation was observed. To minimize emissions, the solution was adjusted to pH 4-5 with sodium acetate buffer. Polysaccharide activation was initiated by adding a sodium metaperiodate solution. To achieve the target level (number of moles of aldehyde per mole of polysaccharide repeating units), meta The amount of sodium periodate added was controlled.

[0300] The activation products of all serotypes except ST-5 are tested with 10 mM potassium phosphate (pH 6.4). Diafiltration is performed against the tangential flow of 5 or 10 kDa NMWCO. Diafiltration was performed on the water using an ultrafiltration membrane. Regarding ST-5, The activation product is dissolved in 10 mM sodium acetate (pH 4.1) in a diafil tray. Then, a diaphragm is used to filter the water using a 5kDa NMWCO tangential flow ultrafiltration membrane. Filtering was performed. Ultrafiltration was carried out at 2–8°C for all serotypes.

[0301] Conjugation of polysaccharides into CRM197 As already stated (WO 2012 / 173876 A1) Pseudomonas • Expression in fluorescein (Pseudomonas fluorescens) The purified CRM197 obtained by this method was filtered using a 5kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration was performed using 2 mM phosphate (pH 7.2) buffer. The solution was filtered through a 0.2 micron filter. The activated polysaccharide was then freeze-dried with water and sucrose. Formulated with 6 mg Pr / mL of CRM197 (CRM197 carrier protein is "Pr"). It was formulated with a sucrose concentration of 1% w / v (also known as) for freeze-drying. The solutions of Ps and CRM197 were individually freeze-dried. 197 materials were individually redissolved in an equal volume of DMSO. For some serotypes, the salts were... Eel additives were added to Ps-DMSO. Target polysaccharide concentration and polysaccharide-to-CRM197 mass. The polysaccharide and CRM197 solutions were mixed so that the ratio was achieved. The polysaccharide-to-CRM197 ratio in the conjugate was selected to be controlled for most In the case of serotypes, a reducing agent such as sodium borohydride is added and conjugate The chemical reaction was carried out at 22°C.

[0302] Final reduction For all serotypes, after the conjugation reaction, a residue such as sodium borohydride is used. The base agent was added and incubated at 22°C. The batch was then mixed with approximately 0.025% (w / v) of the solution. It was diluted at approximately 4°C in 150 mM sodium chloride containing Resorbate-20. Then potassium phosphate buffer was added to neutralize the pH. Several lots were concentrated, and 30 Using a kDa NMWCO tangential flow ultrafiltration membrane, 150 mM sodium chloride, 25 m Diafiltration was performed at approximately 4°C with M potassium phosphate (pH 7).

[0303] Final filtration and product storage Next, each batch is concentrated and filtered using a 300kDa NMWCO tangential flow ultrafiltration membrane. , 150 mM sodium chloride containing 0.015% (w / v) polysorbate 20 Diafiltration was performed at 4°C in 10 mM histidine in pH 7.0. In particular, with regard to ST-5, during the diafiltration process, ST-5 conduit The saturates were collected and incubated with 50 mM sodium bicarbonate (pH 9.3) for 3 hours. Before completing the diafiltration, the ST-5 solution was treated with 1.5M phosphoric acid. It was neutralized with potassium (pH 6.0).

[0304] Each residual batch is filtered through a 0.2 micron filter (0.5 micron filter). (Using a pre-filter), then containing 0.015% (w / v) polysorbate 20. Dilute with an additional 10 mM histidine in 150 mM sodium chloride (pH 7.0). The solution was dissolved, dispensed into aliquots, and frozen at -60°C or below. Detailed information on serotype-specific conjugates was obtained. Details can be found as already described (WO2011 / 100151, WO20 (19 / 139692 and WO2020 / 131763).

[0305] Example 2: Formulation of a pneumococcal conjugate composition Individual pneumococcal polysaccharides produced using various chemicals as described in Example 1 - The carrier protein conjugates are paired with PCV1, PCV21, or PCV24 respectively. It was used in the formulation of monovalent, 21-valent, or 24-valent pneumococcal conjugate compositions.

[0306] PCV1 preparations, which are added to or used as is with CLA-SNE or SNE, are blood It contains pure form 6B, which is aprotic (DMSO) as described in Example 1. ) Conjugated using reductive amination in a solvent, and 4 μg / mL in the vaccine ( 20 mM L-histidine ( Formulated in 150 mM NaCl and 0.1% (w / v) PS-20 at pH 5.8. The reductive amine in a non-protic (DMSO) solvent was performed as described in Example 1. PC manufactured using APA and serotype 6B conjugated using no-conjugation. The V1 vaccine formulation contains 4 μg / mL (w / v) of pneumococcal polysaccharide (PnP) in the vaccine. To achieve the final concentration, use 20 mM L-histidine (pH 5.8) and 150 mM NaC. l and 0.2% (w / v) PS-20 and 250 mg [Al +3 ] / mL (APA Formulation (in terms of morphology).

[0307] PCV21 preparations that are added to or used as is with CLA-SNE or SNE are Serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylated -15B (de-OAc15B), 16F, 17F, 19A, 20, 22F, 23A, 23B It contains 24F, 31, 33F and 35B, which are aprotic solvents (for example, Conjugate to the carrier protein CRM197 using reductive amination in DMSO. Furthermore, 20 mM L-histidine (pH 5.8), 50-150 mM NaCl and 0 Formulated in PS-20 at a concentration of 0.02-0.1%. The vaccine contains 84-168 μg / mL. Add 4-8 μg of each polysaccharide-carrier protein conjugate to achieve the final PnP concentration. Formulated with Streptococcus pneumoniae polysaccharide (PnP) at a concentration of / mL (w / v).

[0308] PCV24 preparations that are added to or used as is with CLA-SNE or SNE are Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24 It contains F, 33F, and 35B, which are present in an aprotic solvent (e.g., DMSO). It is conjugated to the carrier protein CRM197 using reductive amination, and 20m M L-histidine (pH 5.8), 50-150 mM NaCl and 0.02-0. Formulated in 1% PS-20. The final concentration of PnP in the vaccine was 96 μg / mL. To achieve this, each polysaccharide-carrier protein conjugate is administered at 4 μg / mL (w / v) to Streptococcus pneumoniae. It was formulated with polysaccharides (PnP).

[0309] In order to manufacture PCV preparations, the final concentration of pneumococcal polysaccharide (also known as PnP) is ( The required volume of monovalent bulk conjugate needed to obtain w / v is calculated using batch volume and batch volume. The calculation was based on the luc polysaccharide concentration.

[0310] The formulation process involves 20 mM histidine and 0.05-0.15% (w / v) PS-2. The final concentrations of the PnP blend in 0 and 150 mM sodium chloride (pH 5.8) are 2 It consisted of the production of a double batch of conjugate bulk blends.

[0311] Prepare a solution of histidine (pH 5.8), PS-20, and sodium chloride, and formulate it into a liquid. Added to the container. 2-5 individual frozen-stored pneumococcal polysaccharide-carrier protein conjugates The polysaccharide-carrier protein conjugate was thawed at 8°C and then added to a formulation container. When adding to the composting buffer (conjugate blend), use a magnetic stirring rod or magnetic impeller. The container was mixed using [a specific method] to ensure uniformity. After adding all the ingredients and stirring the solution, The conjugate blend is passed through a sterile filter and stored in a container in or without APA. It was collected inside. In some cases, a sterile filter was used to adjust the batch to the target concentration. The substance was chased with 150 mM sodium chloride.

[0312] The formulation was filled into plastic syringes, glass syringes, or vials.

[0313] The PCV13 formulation, a 13-valent pneumococcal conjugate vaccine used herein, It originated from the commercially available PREVNAR13 (registered trademark).

[0314] Example 3: Cationic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa Stable nanoemulsions (S) in and without the presence of -13,16-diene-1-amine NE) Manufacturing of adjuvant-based compounds SNE adjuvants are also known as CLA (13Z,16Z)-N,N-dimethyl- Also known as 3-nonyldocosa-13,16-diene-1-amine or CLX (6Z, 9Z,26Z,29Z)-N,N-dimethylpentatriaconta-6,9,26,29- N,N-dimethyl-1-((1S, also known as tetraen-18-amine or CLY) Catio (2R)-2-octylcyclopropyl)heptadecane-8-amine (Figure 1) SNE can be produced in the presence or absence of cationic lipids. SNE is a cationic lipid, for example, C In the presence of LA (referred to as CLA-SNE; see Table 1) and cationic lipids In the absence of (referred to as SNE; see Table 2), the three stabilizing components are squa Len, sorbitan trioleate (SPAN-85) and polysorbate-20 (PS -20) is a multi-component emulsion formulation. This formulation is made of cationic lipids (used (In the case of) squalene, SPAN-85 and PS-20 or similar (e.g., surfactant The product was manufactured by combining and mixing the components (agents, oils, and solubilizers) (Table 1 and Figure 2). ). After mixing and blending, add histidine buffer and the initial emulsion components. Mixed. As described below, the blended emulsion components were first subjected to rough homogenization. Next, the mixture was subjected to a fine homogenization process. The resulting formulation was then subjected to a final 0.2 μm filtration step. To produce an emulsion system with desirable properties, several steps are taken in each process. Process parameters, such as the order of addition, mixing time, pH, temperature, concentration of components, homogenization, and micronization. Microfluidization was controlled.

[0315] [Table 1]

[0316] [Table 2]

[0317] Manufacturing of pharmaceutical products The stable emulsion of squalene and solubilizer preparation (referred to as the oil phase) is composed of squalene. It was prepared by adding SPAN-85, PS-20, and CLA to a container. Next The oil phase was then mixed by magnetic stirring at 100-1000 RPM for 10-120 minutes. After mixing these components, the aqueous phase consisting of 20 mM histidine (pH 5.8) is subjected to magnetic stirring. The mixture was slowly added to the oil phase while mixing with a stirring rod. Then the mixture was mixed again for 1 hour. did.

[0318] Coarse homogenization Next, the rotor stator homogenizer is used at ambient temperature to mix the oil phase and the water phase. The mixture (referred to as pre-homogenized emulsion or PHE) is homogenized and its size is reduced. A crude emulsion was formed. The tip of the homogenizer arm was submerged in the PHE, and the formulation was carried out. It was kept in place near the bottom of the container and operated at 6-10kRPM for 5-15 minutes. Roseth is a homogeneous microemulsion of squalene emulsion particles in the diameter range of 4-20 μm. A (ME) suspension was given. This was to generate a stable nanoemulsion (SNE). For example, additional size by microfluidization in a high-pressure homogenizer. It was suitable for reduction.

[0319] Fine homogenization for generating stable nanoemulsions (SNEs) To generate stable nanometer-sized emulsion particles after rough homogenization The emulsion is further processed using a high-pressure homogenizer / microfluidizer. For example, microfluidics (low-volume microfluidics) Microfluidizer (registered trademark), GEA Group Pa ndaPlus 2000 or Bee International, NanoDeB We introduced ME into a high-pressure homogenizer like EE and established a recirculation loop. High-pressure homogenization To neutralize the heat generated, a temperature control unit with a set temperature of 5°C is supplied. A counterflow heat exchanger is included in the recirculation loop. To manufacture the particle, select 20 kPSI as the operating setting for this process step. The high-pressure homogenizer provides a constant and unchangeable flow rate through an established recirculation loop. It operates. Using this measured flow rate and processed ME volume, the entire formulation passes through the recirculation loop once. We calculated the theoretical time required to pass through. Taking this calculated single pass time into account, normally The high-pressure homogenizer is operated until at least 10 desired passes are reached, and the SNE Alternatively, I obtained either a CLA-SNE.

[0320] filtration After formulation, the SNE or CLA-SNE is passed through a 0.8 / 0.2 μm PES filter. It was allowed to pass through. Considering the optimal mass yield and particle stability through filtration, the particles passed through the filter. I selected a flux of 42 LMH.

[0321] Using Malvern Panalytical Ltd.'s MS3000 system Using laser diffraction or static light scattering (SLS) techniques, the body of nanoemulsions during manufacturing is analyzed. The product-weighted size distribution was measured. This data was then analyzed to generate scattering patterns. Particle size was calculated. Pre-homogenized emulsion (PHE), microemulsion (M Sample fractions of E) and stable nanoemulsions (SNE) were obtained. Prepare the formulation to achieve a 3% obscuration, 5m Dilute in M ​​histidine (pH 5.8) and 2.5 mM NaCl buffer, SL S was performed and data was collected under a 1200 RPM recirculation. The sample dataset was then used in the dataset. Data was collected with a 30-second scan per set. Each step in the CLA-SNE formulation process. Three datasets from the following sources are summarized in Figure 3: 20 mM histidine (pH 5.8) During processing and storage in polymer containers (e.g., plastic), the fur is used. Although it was a perfectly suitable formulation for the stability of the product, during storage in glass, the glass surface... Nonspecific absorption of CLA-SNE or SNE into the surfactant / solubilizer was observed. Screening was conducted to evaluate buffers and salts, and multiple formulations were identified as stabilized formulations. Selected 20 mM histidine, 0.05% PS-20, and 75 mM NaCl The selected formulation demonstrated that this stability problem was successfully resolved (data not shown). (shown).

[0322] Example 4: Preparation of a stable nanoemulsion (SNE) adjuvant system, and the process of preparing SNE CLA or cationic lipids as free bases immediately after microfluidization (13 Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine Addition PS-20 and sorbitan triolet are blended in histidine (pH 5.8) buffer. CLA into nanoemulsion particles containing art (SPAN-85) and squalene Regarding uptake, two formulation processes were evaluated. The first process (referred to as Process 1) In the case described in Example 3, SNE was manufactured using the process described in Example 3. In process 2 (referred to as process 2), PS-20 and sorbitan triolea are used. Only t(SPAN-85) and squalene were combined and mixed. Then, histidine buffer is added, and the first emulsion component (PS-20, sorbita) is added. Mixed with (trioleate and squalene). As described in Example 3, The blended emulsion components are first subjected to coarse homogenization to form microemulsions ME. This is used to generate a nanoemulsion (NE) which is then subjected to microfluidization. It was generated. In a separate glass container, 0.25 mg / mL of CLA was dissolved at room temperature in 100% aqueous solution. It was dissolved in tanol. Then, a sufficient volume of this CLA was added to obtain the desired final CLA concentration. Add PS-20 and sorbitan to the ethanol solution in histidine buffer (pH 5.8). Add rioleate and squalene to SNE, then mix at room temperature for 60 minutes. Next, after incubation, the formulation was mixed with 10 mL of buffer in 500 mL of buffer. The sample ratio is 4 for 5 mM histidine and 2.5 mM NaCl (pH 5.8). Dialysis was performed overnight at °C, with two buffer changes. Then, using UPLC-CAD, Two processed emulsions (process 1 and 2) are used for CLA incorporation into SNE. The following tests were conducted. The results showed that when using process 2, the target uptake rate of each formulation was ( This indicates that a significant amount of CLA was not taken up compared to (w / w)%). This indicates that the successful incorporation and stability of CLA in nanoemulsions is due to a process Figure 4 indicates that S1 is preferable.

[0323] Example 5: Preparation of cationic lipid-containing LNP adjuvant CLA-LNP consists of four components, namely one cationic lipid [referred to as CLA]. In Figure 1, the preferred cationic lipid is CLA, i.e., (13Z,16Z) -N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine Cholesterol, distearoylphosphatidylcholine (DSPC), and ePE This is a multi-component LNP preparation consisting of G-DMG.

[0324] The relative target molar values ​​for the final CLA-LNP formulation regarding lipid components are 58% CLA and 30% LNP. It is % cholesterol, 2% ePEG2000-DMG and 10% DSPC. Table 3).

[0325] [Table 3]

[0326] The manufacturing process for CLA-LNP consists of the following five steps: 1) Lipid mixture and dilute 1) Preparation of citrate A solution; 2) Formation of LNP by mixing; 3) Ultrafiltration; 4) Bioba -Density reduction filtration; and 5) Sterile filtration and vial filling.

[0327] Preparation of a solution of lipid mixture and diluted citrate A The lipid components were weighed, combined, dissolved in ethanol, and then sterile filtered to form a lipid mixture. I obtained the substance. Dilute citrate A (20 mM citrate, pH 5.0) with sterile water in a 1:1 ratio. The solution was dissolved to obtain diluted citrate A (DCA).

[0328] LNP formation by T-mixing Next, the lipid mixture and DCA were mixed together at the adjacent end of the T-tube mixer. The flow exiting the T mixing device is mixed with 20 mM citrate and 300 mM NaCl (pH 6.0). Dilute immediately 1:1 with 1×Dulbeccio phosphate buffered physiological salt, then mix the product with 1×Dulbeccio phosphate buffered physiological salt. The LNPs were then diluted again with water at a 1:1 ratio and collected as formed LNPs. The bodies were incubated at ambient temperature for 30 minutes, then kept at 4°C overnight.

[0329] Ultrafiltration Next, the LNP intermediate is subjected to ultrafiltration in 500 kDa NMWCO, and the material is approximately 1 The material is concentrated to 0 times its original concentration, and the material is mixed with 20 mM Tris and 10% (w / v) sucrose (pH Diafiltration was performed on 7.5). After diafiltration, the final A final concentration process was performed to obtain the target concentration.

[0330] Bioburden reduction filtration Next, the adjuvant bulk was filtered through a 0.45 μm cellulose acetate (CA) filter. Pre-filter with a 0.2 μm CA bioburden reduction filter and store frozen at -70°C. did.

[0331] Sterile filtration and vial filling The frozen adjuvant bulk was thawed in a water bath controlled at 25±3°C. The adjuvant bulk was then processed using 0.45 μm polyvinylidene fluoride (PVDF) bio The material is passed through a baden reduction filter and a 0.22 μm PVDF sterile grade filter. So, it was recovered. Next, the filtered adjuvant bulk was treated with 20 mM Tris, 10% (w / v) The LNP adjuvant was diluted with sucrose (pH 7.5) to the target concentration. Then, This diluted final bulk adjuvant is filled into glass vials and stored at -70°C. Ta.

[0332] Example 6: PCV1 immunogenicity in mice: Evaluation of adjuvant systems Young female BALB / c mice (6-8 weeks old, n=10 / group) were given various adjuvants (see table). 4) 0.1 mL of PCV1 containing the above, administered intramuscularly (I M) was immunized. 0.08 μg of PnPs (conjugates CRM197) per immunization. The mice were administered modified 6B). The mice were trained in relation to any signs of disease or distress. Vaccine-related adverse events were observed at least daily by trained animal care staff. Since no adverse reactions were observed, the vaccine formulation in mice is considered safe and well-tolerated. All animal experiments were conducted by the National Institutes of Health (NIH). Guidelines for the Management and Use of Laboratory Animals (TeS of Health) (for Care and Use of Laboratory Animals) The study was conducted in strict adherence to the recommended procedures. The mouse experiment protocol was developed by Merck & Co. Animal Management and Use Committee at Kenilworth, Inc. (Kenilworth, NJ, USA) (Institutional Animal Care and Use Commi. It was approved by ttee.

[0333] [Table 4]

[0334] Mouse serum was evaluated for IgG immunogenicity using ELISA, and its anti-6B IgG activity was assessed. The titer was evaluated. Functional antibodies were tested at the University of Alabama (UAB). Owned and licensed by the Research Foundation Opsotiter® 3 software and UAB Pneumococc al Reference Laboratory(University of Al abama Reference Laboratory at Birmingham Bacterial Respiratory Pathogen Reference Opsonic based on the already described protocol available from the e Library Determined by phagocytosis assay (OPA) (Caro-Aguilar I. et al., V accine(2017)35(6):865-72 and Burton RL Nahm MHClin. Vaccine Immunol. (2006) 13 (See (9):1004-9). As shown in Figure 5A, PCV1 immunization In vaccines formulated in the presence and absence of APA, serotype 6B (ST) is present. Antibody titers against -6B) were generated in BALB / c mice. CLA-SNE (dose Formulated with 0.08, 8, or 80 mg of CLA or SNE adjuvant per dose. ST-6B was found to be immunogenic in mice, either alone or in combination with APA. Compared to formulated ST-6B, it exhibits higher immunogenicity after the third dose. BALB / immunized with ST-6B formulated in the presence and absence of APA In mouse c, an anti-6B functional antibody titer was generated (Figure 5B).

[0335] Example 7: Immunogenicity of PCV24 in adult rhesus monkeys PCV24 (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac-15B, 18C, 19A, 19F, 22F, 23B Each of the 23F, 24F, 33F, and 35B units is individually connected to CRM197. The (processed) substances were evaluated in an adult rhesus monkey immunogenicity model. Day 0 and On the 28th, PCV24 containing APA, or PCV24 containing SNE or SNE(CL) PC containing CLA formulated as A-SNE or LNP (CLA-LNP) In V24 (Table 5), rhesus monkeys were immunized intramuscularly. 0.1 mL of immunization was administered per unit volume. PCV24 was administered with 0.4 μg PnPs. Before the start of the study (before immunization, day 0) Serum samples were collected on day 14 (PD1) and day 42 (PD2).

[0336] [Table 5]

[0337] To evaluate the serotype-specific IgG immunogenicity response in 24-valent vaccines, multiplexed electrons We developed a chemiluminescence (ECL) assay. This assay was developed by Marchese et al. and S. Kinner et al. (Marchese RD et al., Clin. Vaccine Immu) nol. (2009) 16(3):387-96 and Skinner, JM et al., V This is described in accine (2011) 29(48):8870-8876). Developed for use in rhesus monkey serum based on previous assays. MesoSca le Discovery(MesoScale Diagnostics,LLC,G The technology developed by Aithersburg (a division of MD) is 96 well play Pneumococcal serotype polysaccharides (1, 3, 4, 5, 6A, 6) coated on spots within the t B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, OA removal c-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35 B) and a SULFO-TAG® labeled antibody that emits light upon electrochemical stimulation It is used to test rhesus monkey serum samples with SULFO-TAG(trademark) labeled anti-human IgG. It was used as a secondary antibody for testing. Using the logarithmic scale of ECL and dilution, the cuts were made. The endpoint dilution force is defined as the reciprocal of the linear interpolation dilution corresponding to the tooff value (control ECL signal). The value was calculated. The last two or three sample curves that are completely above the cutoff line. Linear extrapolation (log-log scale) using intercepts and slopes of ECL assay data points. Next, the titer was extrapolated for samples exceeding the maximum dilution considered. All titers are The sample curve was obtained by inversely transforming the linear extrapolation dilution. If located below, all data analysis and titers in Figures 6A and 6B will be used. 100 was used. Functional antibodies were obtained from the University of Alabama (UAB). )Owned and licensed by the Research Foundation The Opsotiter® 3 software and UAB Pneumococ cal Reference Laboratory(University of A labama Reference Laboratory at Birmingha m Bacterial Respiratory Pathogen Referen Multiplexing based on already documented protocols available from the ce Library. Determined by opsonin phagocytosis assay (MOPA) (Caro-Aguilar I. et al., Vaccine (2017) 35(6):865-72 and Burton RL and Nahm MHClin. Vaccine Immunol. (20 See 06)13(9):1004-9).

[0338] 1200 μg / mL CLA-SNE (25 mg / mL squalene, 5.0 mg / m³) PCV24, which contains L-PS-20 and 5.0 mg / mL SPAN-85, is for adult red eggs. It was found to be immunogenic in ghezal, and compared to PCV24 containing APA, Higher immunogenicity was observed after the first and second doses (Figure 6A). 120 μg PCV24, which contains CLA-SNE at dose levels, is effective after the first dose (PD1) and the second dose. After 2 doses (PD2), it was found to be equivalent to or better than PCV24 containing APA (solid line). It exhibits good immunogenicity. In PD2, there are two dose levels of CLA (indicated by circles) Contains 120 μg of CLA-SNE (or 80 μg, indicated by a triangle). PCV24 is either PCV24 containing APA (solid line) or CLA-LNP (120 μg Compared to PCV24 containing (displayed as a square), it exhibits equivalent or better immunogenicity. It brings about. (Figure 6B).

[0339] These same formulations contain functional antibodies that kill the vaccine-type bacterial strain at all time points in the test. Generated (Figures 7A-7M), CLA-SNE, SNE alone, and CLA-LNP again, Compared to PCV24 formulated with APA, the PCV24 response was enhanced. One exception was 23B. Furthermore, their high pneumococcal preliminary immunization prevented a booster immune response after vaccination. The study time was tested in MOPA as pooled samples or individual samples. (Figures 7A-7M). No significant difference in OPA titer was observed between PD1 and PD2.

[0340] The serum of adult rhesus monkeys immunized with PCV24 was used to test for other Streptococcus pneumoniae. Cross-reactivity to Bacteria was evaluated. Serum of rhesus monkeys immunized with PCV24. This is an exchange for serotypes 6C (Figures 6A, 6B, and 7D) and 15B (Figures 6A and 6B). Differential reactivity was observed. Cross-reactivity to 6C was observed as polysaccharide condyloma as part of polyvalent PCV24. This may be due to immunization with 6A-CRM197 (Cooper D, Yu X, Sidhu M, Nahm MH, Fernsten P, Jansen KU). The 13-valent pneumococcal conjugate vaccine (PCV13) is stress-free in humans. Cross-functional opsonin phagocytosis against *Ptococcus pneumoniae* serotypes 6C and 7A It induces a loss response (Vaccine.2011;29:7207-11). Similarly, polyvalent Immunization with polysaccharide conjugate-de-O-Ac-15B-CRM197 as part of PCV This resulted in cross-reactivity to serotype 15C (Rajam et al., Clinical a nd Vaccine Immunology,2007,14(9):1223-12 27).

[0341] Example 8: Study on the immunogenicity of PCV24 in infant rhesus monkeys (IRM). PCV24 (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, de-O-Ac-15B, 18C, 19A, 19F, 22F, 23B Each of the 23F, 24F, 33F, and 35B units is individually connected to CRM197. The (formulated) and adjuvant formulations are manufactured as described in the above examples. IRM (infant rhesus macaques, n=5 / group) was conducted on day 0, day 28, and day 56. As shown in Table 6 below, immunization was induced in the muscles with 0.1 mL of vaccine. Before the start (before) and also on the 14th day (PD1), the 42nd day (PD2) and the 70th day (PD 3) Serum was collected. IRM was performed twice a day by trained animal care staff. Observed in relation to any signs of illness or distress. No vaccine-related adverse events were observed. Because no such issues were observed, the vaccine formulation was deemed safe and well-tolerated by the IRM. .

[0342] [Table 6]

[0343] To evaluate the serotype-specific IgG immunogenicity response in a 24-valent vaccine, the above-mentioned method We developed a multiplexed electrochemiluminescence (ECL) assay for use in cages. ECL and dilution Using a logarithmic scale, linear interpolation is performed on the cutoff value (control ECL signal). The endpoint titer was calculated as the reciprocal of the determinant. Sample music located exactly above the cutoff line. Out-of-line analysis using the intercept and slope of the last two or three ECL assay data points of the line. Based on the interpolation (logarithmic-logarithmic scale), for samples exceeding the maximum dilution examined, the titer The value was extrapolated. Then, the titer was obtained by inversely transforming the linear extrapolated dilution. Sample If the curve is located completely below the cutoff line, then in all data analysis and drawings... A titer of 100 was used.

[0344] As shown in Figure 8A, after the second dose (day 42), P containing CLA-LNP CV24 provides equivalent or better immunity compared to PCV24 (circled), which contains APA. It causes adverse effects. CLA-SNE (2.5 mg of squalene per 0.1 mL dose, 0 295 μg of CLA in the presence of 0.25 mg of PS-20 and 0.25 mg of SPAN-85 (Indicated by a triangle) or CLA-SNE (0.5 mg per 0.1 mL dose) 29 in the presence of squalene, 0.05 mg of PS-20, and 0.05 mg of SPAN-85 PCV24, which contains 5 μg of CLA (shown in diamond shape), is a PC containing APA. It exhibits comparable or better immunogenicity compared to V24. PCV13 and PCV2 Except for ST5, 4 / APA exhibits equivalent immunogenicity against serotype (ST) in common. (Square). ST5 shows higher immunogenicity with respect to PCV24 / APA.

[0345] CLA-SNE (2.5 mg squalene and 0.25 mg P per 0.1 mL dose) 60 μg of CLA in the presence of S-20 and 0.25 mg of SPAN-85; indicated by the circle. (is present), CLA-SNE (2.5 mg squalene per 0.1 mL dose, 0.25 mg 120 μg of CLA in the presence of 0.25 mg of PS-20 and 0.25 mg of SPAN-85; triangular (as indicated) or CLA-SNE (2.5 mL of squalane per 0.1 mL dose) 295 μg in the presence of 0.25 mg of PS-20 and 0.25 mg of SPAN-85 PCV24 containing CLA (shown in diamond shape) contains PD1 (Figure 8B), PD2 (Figure 8B). In 8C) and PD3 (Figure 8D), comparable results were obtained compared to PCV24 containing APA. Or it results in better immunogenicity. Notably, 0.0 per 0.1 mL dose. 8mg squalene, 0.008mg PS-20, 0.008mg SPAN-85 PCV24, which contains CLA-SNE at a dose of 120 μg of CLA, is administered in 0.1 mL doses. Each dose contains 0.5 mg of squalene, 0.05 mg of PS-20, and 0.05 mg of SPAN- PCV24 containing 85 and 120 μg (CLA) of CLA-SNE, or 0. Each 1 mL dose contains 2.5 mg of squalene, 0.25 mg of PS-20, and 0.24 mg of [unclear]. Compared to PCV containing CLA-SNE with SPAN-85 and CLA295μg It exhibits equivalent immunogenicity (data not shown).

[0346] Example 9: PCV24 defense against challenge in mice Young female Swiss Webster mice (6-8 weeks old, n (=10 / group) 0.1 mL of 24-valent Streptococcus pneumoniae conjugate was administered on day 0, day 14, and day 28. The patient was immunized intramuscularly (IM) with the jugate vaccine (PCV24). PCV24 was administered. PCV24 is a PnPs(1 , 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, De-O-Ac-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33 F and 35B) were administered at 0.4 μg. PCV24 was as described in Table 7. It was formulated using several adjuvants. The mice were one of Observations for signs of illness or distress were made daily by trained animal care staff. No vaccine-related adverse events were observed, therefore the vaccine formulation is safe in mice. Furthermore, it was considered highly tolerable. All animal studies were conducted at the National Institutes of Health (NIT). Regarding the management and use of laboratory animals at the National Institutes of Health Guidelines for Care and Use of Laboratories The mouse experiment protocol was conducted in strict adherence to the recommendations of (y Animals). Implementation at Merck & Co., Inc. (Kenilworth, NJ, USA) Institutional Animal Care and Use Committee It was approved by the Use Committee.

[0347] On day 53, mice were anesthetized with isoflurane and given Streptococcus pneumoniae serum. I performed an intratracheal challenge with type 24F. To put it simply, it was a Streptococcus pneumoniae. The logarithmic culture of E was centrifuged, washed, and suspended in sterile PBS. 0.1 mL of PBS 4.6 x 10 inside 6 Streptococcus pneumoniae of cfu, erected by the incisors. It was placed in the throat of a mouse suspended in a cage. The tongue was slowly pulled outwards, covering the nostrils. Therefore, inhalation of the bacteria was induced. The weight of the mice was measured daily, and weight loss was measured as 2% of the starting weight. If the percentage exceeded 0%, the animals were euthanized. Bacteremia was assessed 24 hours after the challenge. Blood samples were taken at 48 and 72 hours. The mice were given any disease or suffering. Regarding signs of pain, they should be observed at least twice a day by trained animal care staff. Ta.

[0348] Includes adjuvant-added vaccines (CLA-LNP, CLA-SNE, or SNE alone). Mice immunized with PCV24 were protected from intratracheal challenge by serotype 24F. Figure 9). All mice immunized with the PCV24 formulation containing adjuvant were challenged. The survival rate was 100% on day 7 after treatment, compared to the survival rate of untreated (naive) mice. The percentage was 10%. This data shows that PCV24 containing adjuvant formulations was serotype 24 This demonstrates that we were able to defend the mouse from the IT challenge in F.

[0349] [Table 7]

[0350] Example 10: Physical stability of nanoemulsion formulations using NTA and DLS The effects of time and temperature on this As shown in Figure 10, the nanoemuls prepared as described in the above examples. To evaluate the stability of the ion system (CLA-SNE or SNE), nanoparticle tracking analysis is performed. (NTA) was used. This technique directly tracks particles to estimate their size and concentration. Collect a video of a group of particles as they move due to Brownian motion. The 635nm laser in S1 focuses an 80mm red laser beam through a liquid sample. This causes the particles to be illuminated as rapidly diffusing points of light. The CCD camera captures each individual To track the motion of the irradiated particles over time, video is recorded at 30 frames per second. Tem Software identifies the center of each individual particle from the video and crosses it independently. The distance is tracked to determine the mean squared displacement. Raw data collected from the entire video is divided Until analysis is performed, this tracking will be the same for all particles in the sample population in each frame. This was done by simultaneously measuring the mean square displacement of all the individual particles being tracked. , its diffusion coefficient (Dt) and spherical equivalent hydrodynamic radius (spherical equi The valent hydrodynamic radius (rh) is used in Stokes-A This was determined by applying Instein's equations. The software then uses this accumulation. The data is displayed as particle size and concentration distribution. In addition to particle size and concentration, Raw data information regarding the intensity or brightness of individual particles was also collected. The data was then compiled into a file. Fitting, as particle strength relative to particle size and particle concentration relative to particle size Plot each particle individually, then compare the particle size, concentration, and intensity of all particle populations. The data was plotted on a dimensional contour plot.

[0351] Nanoemulsion formulations (CLA-SNE or SNE) at 4°C, 25°C and 37°C Even after exposure for up to one month, the particle concentration of the nanoemulsion remained unchanged in the evaluation using NTA. No significant changes were observed in the size distribution (Figure 10).

[0352] Because nanoemulsions tend to aggregate within a particle size range of 10-1000 nm, DLS is... This provides an appropriate stability indication technology for evaluating and quantifying aggregation phenomena. To evaluate the stability of nanoemulsion systems manufactured as described, dynamic light scattering (D) is used. The average particle size distribution was measured using LS. The DLS instrument uses a laser to measure the distribution in the solution. Particles are irradiated, and then the change in the intensity of scattered light over time as a result of Brownian motion is examined. The correlation between the intensity at time zero and the scattered light intensity over time is an exponential decay curve. This yields a linear or correlation function. The decay rate in the correlation function with respect to time is greater for larger particles. The process is much faster for smaller particles, and this forms the basis for calculating particle size. Exposure of nanoemulsions to 4°C, 25°C, or 37°C for up to one month did not affect the nanoemulsions. No changes in the size distribution of the ions were observed by DLS (Figure 11). CLA-S For NE or SNE, the average Z value remained approximately 110nm to 180nm.

[0353] Example 11: Chemical stability of nanoemulsion formulations when using UPLC-CAD The effects of time and temperature on this The chemical stability of the nanoemulsion system manufactured as described in the above examples is To evaluate this, we used ultra-high performance liquid chromatography (UP) combined with a charged particle detector. Using LC-CAD, CLA (Chemical Latitude Amount) during storage for one month at 4°C, 25°C, and 37°C. The stability and squalene concentration of CLA-SNE (only) were measured. 4 nanoemulsions were used. Even when exposed to temperatures of 25°C, 25°C, and 37°C for up to one month, the CLA in SNE (Figure 12A) also The concentration of squalene (Figure 12B) was not affected. Furthermore, UPLC-CAD was squalene The formation of degradation products by the chemical decomposition of allene or CLA can be quantified. SNE or CL Even when the A-SNE adjuvant system was exposed to high temperatures, no detectable resolution peaks were observed. This is because CLA-SNE and SNE adjuvant-based squalene and CLA components This demonstrates excellent thermal stability (data not shown).

[0354] Example 12: Stable emulsion system for the stability of pneumococcal conjugate vaccine ( Impact of + / -CLA) Individual Streptococcus pneumoniae strains prepared using a reductive amination solvent (aprotic DMSO) 192 μg of sugar-carrier protein conjugate, as described in the above examples. It was used in formulation of PCV24 at a concentration of / mL.

[0355] The PCV24 composition is mixed with various adjuvant systems listed in Table 8 in a glass container. The mixture was placed together and left at 4°C for up to 30 days. The formulation showed good stability, and CLA-SNE( 1.2 mg / mL CLA-SNE [6.5 mg / mL squalene, 0.65 mg / mL] L PS-20, 0.65 mg / mL SPAN-85, or 1.2 mg / mL CL A-SNE [1.2 mg / mL squalene, 0.12 mg / mL PS-20, 0.1 [2 mg / mL SPAN-85]) or SNE ([6.5 mg squalene, 0.65 [0.65 mg of PS-20, 0.65 mg of SPAN-85] or [0.4 mg of squalene, [0.04 mg PS-20, 0.04 mg SPAN-85]) are fluorescence-based ELI When using the SA assay, dose stability of the pneumococcal polysaccharide-carrier protein conjugate was achieved. It did not affect sexuality (Figures 13A-13D).

[0356] [Table 8]

[0357] Example 13: PCV21 immunogenicity in adult rhesus monkeys Also, PCV21 (serotypes 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15 A, de-O-acetylated-15B (de-OAc15B), 16F, 17F, 19A, 20, 2 2F, 23A, 23B, 24F, 31, 33F, and 35B are each individually CRM1 (conjugated to 97) as described in the above example, adult red The immunogenicity was evaluated in a rhesus monkey model. Rhesus monkeys were subjected to PC on day 0 and day 28. P is formulated with V21 alone or in combination with CLA (CLA-SNE) formulated as SNE. In CV21 (Table 9), immunization occurred in the muscle. 1.0 in a volume of 0.25 mL per immunization. PCV21 was administered via μg of PnPs. Before the start of the study (before immunization, day 0) and at day 0 Serum samples were collected on the 14th (PD1), the 28th (PD1), and the 42nd (PD2).

[0358] [Table 9]

[0359] To evaluate the serotype-specific IgG immunogenicity response in 21-valent vaccines, multiplexed electrons We developed a chemiluminescence (ECL) assay. This assay was developed by Marchese et al. and S. Kinner et al. (Marchese RD et al., Clin. Vaccine Immu) nol. (2009) 16(3):387-96 and Skinner, JM et al., V This is described in accine (2011) 29(48):8870-8876). Developed for use in rhesus monkey serum based on previous assays. MesoSca le Discovery(MesoScale Diagnostics,LLC,G The technology developed by Aithersburg (a division of MD) is 96 well play Pneumococcal serotype polysaccharides (3, 6A, 6C, 7F, 8) coated on the spots within the t , 9N, 10A, 11A, 12F, 15A, 15B, OAc15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F and 35B), and This utilizes SULFO-TAG® labeled antibodies that emit light upon electrochemical stimulation. LFO-TAG® labeled anti-human IgG for testing rhesus monkey serum samples It was used as a secondary antibody. In Figures 14A, 14B, and 14C, the IgG concentration is shown as a reference serum standard. Interpolation was performed from quasi-007sp. Functional antibodies were obtained from the University of Alabam Owned by the (UAB) Research Foundation and licensed The Opsotiter® 3 software and UAB Pneu were provided under license. mococcal Reference Laboratory (University of Alabama Reference Laboratory at Birm ingham Bacterial Respiratory Pathogen Re Based on already documented protocols available from the reference library. This was determined by the multiplex opsonin phagocytosis assay (MOPA) (Caro-Agu ilar I. et al., Vaccine (2017) 35(6):865-72 and Bu rton RL and Nahm MHClin.Vaccine Immuno See l.(2006)13(9):1004-9).

[0360] 1.2 mg / mL CLA-SNE (1.2 mg / mL squalene, 0.12 mg / PCV21, which contains 0.12 mg / mL of PS-20 and 0.12 mg / mL of SPAN-85, is for adults. It was found to be immunogenic in rhesus monkeys and was formulated in the absence of an adjuvant. Compared to PCV21, the results after the first dose (Day 14 - Figure 14A; Day 28 - Figure 14B) were Furthermore, higher immunogenicity was observed after the second dose (day 42; Figure 14C) (Figure 6A). 3 PCV21, which contains CLA-SNE at a dose level of 00 μg, shows the following effects after the first dose (PD1): And after the second dose (PD2), compared to PCV21 formulated in the absence of adjuvant It provides comparable or better immunogenicity.

[0361] These same formulations contain functional antibodies that kill the vaccine-type bacterial strain at all time points in the test. When generated, CLA-SNE again shows a PCV21 response compared to PCV21 formulated alone. The study was enhanced. All study time points were treated as pooled samples or individual samples of MOPA. The experiment was conducted in [location]. No significant difference in OPA titer was observed between PD1 and PD2. (Data hidden).

[0362] The serum of adult rhesus monkeys immunized with PCV21 was used to test for other Streptococcus pneumoniae. Cross-reactivity to Bacteria was evaluated. Serum of rhesus monkeys immunized with PCV21. This includes serotype 6C (Figures 14A, 14B, and 14C) and 15B (Figures 14A, 14B, and Cross-reactivity with 14C was observed. Cross-reactivity with 6C was observed in part of polyvalent PCV24. This may be due to immunization with the polysaccharide conjugate 6A-CRM197. (Cooper D, Yu X, Sidhu M, Nahm MH, Fernsten P. Jansen KU). The 13-valent pneumococcal conjugate vaccine (PCV13) is Cross-function in humans against Streptococcus pneumoniae serotypes 6C and 7A It induces an opsonin-mediated phagocytic-killing response (Vaccine.2011;29:7207-1 1). Similarly, polysaccharide conjugate detached O-Ac-15B-CR as part of polyvalent PCV Immunization with M197 (de-OAc15B-CRM197) is cross-reactive against serotype 15C. (Rajam et al., Clinical and Vaccine Immunity) nology, 2007, 14(9):1223-1227).

[0363] Example 14: Microfluidic nanoemulsion self-assembly Cationic lipids (13Z,16Z)-N,N-dimethyl-3-nonyldocor (MNS) Stable nanoemulsions in and without the presence of 13,16-diene-1-amine SNE) Manufacturing of adjuvant-based materials

[0364] Stable nanoemulsion adjuvants are ionizable chemicals, also known as CLA (Figure 1). Thionic lipid (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16- It is produced in the presence and absence of diene-1-amine. To produce SNE, The microfluidic nanoemulsion self-assembly (MNS) process is used. SNE is CL In the presence of A (referred to as CLA-SNE; Table 10) or in the absence of CLA (referred to as SNE) (Table 11), three stabilizing components, namely squalene, sorbitan trioleate A multi-component emulsion consisting of (SPAN-85) and polysorbate-20 (PS-20). It is a stimulant formulation. Biophysical properties of SNE / CLA-SNE formulations manufactured by MNS. (For example, particle size, chemical composition) and stability are as described in Example 3 of the SNE. This is very similar to the high-pressure micro-homogenization process for the manufacture of CLA-SNE formulations. Essentially, the microfluidic nanoemulsion self-assembly described in this embodiment (M The NS process is a modern approach to producing stable nanoemulsion (SNE) adjuvant systems. This is a replacement process. The nanoparticle self-assembly process described in this embodiment is called "microphone This was carried out using a "Rofluidix" ethanol / aqueous mixing apparatus. However, as described in the present invention The ethanol / water flow nanoparticle self-assembly process described is called "microfluidics (microf (Ruydic) Not limited by mixing. A larger volume of hydrophobic solvent stream and aqueous solution The mixing can be achieved using the Tee mixing process outlined in Example 4. ru.

[0365] SNE MNS formulations generally contain cationic lipids, squalene, SPAN-85, and By dissolving PS-20 at the target concentration in a suitable non-aqueous solvent such as ethanol It can be manufactured. The self-assembly procedure involves a flow of hydrophobic emulsion components dissolved in ethanol. This includes combining the flow of the aqueous emulsion solution. Consequently, hydrophobic molecules (i.e., cationic lipids, squalene, SPAN-85 and P) S-20) interacts with the aqueous solvent. Then, as described below, the molecule becomes nano-sized particles. It assembles into an emulsion. After the formation of the self-assembling emulsion of nanoparticles, several appropriate By a reasonable method, residual ethanol can be removed from a stable squalene emulsion. It is possible. In this example, by overnight dialysis using an aqueous buffer, The tanol content was reduced to less than 0.1% (w / v). The resulting SNE formulation was processed with a pore size of 0.2 Sterilized by filtration with a μm sterile filter. SNE with desirable properties To generate the juvant system, several process parameters are required in each step, for example The order of addition, mixing time, temperature, concentration of non-aqueous components, concentration of aqueous buffer components, aqueous pH The mixing ratio of non-aqueous and aqueous solutions, the total flow rate, and the waste volume were controlled.

[0366] [Table 10]

[0367] [Table 11]

[0368] Manufacturing of pharmaceutical products using microfluidic nanoemulsion self-assembly In this example, for biophysical characterization, 20 mM histidine (pH) 5.8) By the microfluidic nanoemulsion self-assembly method, one SNE formulation And four CLA-SNE formulations were manufactured. The self-assembling nanoemulsion process was 1 5 mg / mL squalene, 1.5 mg / mL SPAN-85 and 1.5 mg / mL The process begins by completely dissolving the PS-20 in ethanol. Each solution contains 0.75, 1.5, 5.0, or 15.0 mg CLA / mL of CLA. It also contained CLA / Square. Therefore, the initial "target" for all five formulations was CLA / Square. Len(w / w)% is 0.0, 5.0, 10.0, 33.3 and 100(w / w)% C The solution is LA / squalene. The aqueous buffer for all of this formulation is 20 mM at pH 5.8. It was histidine. Precision NanoSystems, Inc. (Va (Ncouver, BC, Canada) Benchtop NanoAssemblr (Trademark) Using the apparatus, the aforementioned one SNE and four CLA-SNE adjuvant nanoe The marshals self-assembled.

[0369] Self-assembling squalene nanoparticle formulations were manufactured by the following method: In a 1 mL syringe, ethanol A mixture of hydrophobic compounds slightly over 0.7 mL dissolved in ol was filled, while a 3 mL syringe was used. Each container was filled with slightly more than 1.4 mL of 20 mM histidine (pH 5.8) aqueous buffer. After loading the appropriate amount of solution into the syringe, use NanoAssembl It was attached to the r(trademark) device. The following microfluidic mixing parameters were set in NanoAsse Programmed within mblr(trademark): a) Total volume = 2 mL, b) Flow ratio = 2:1 (water to Ethanol), c) Total flow rate = 12 mL / min, d) Starting waste volume = 0.25 mL, e) Final waste volume = 0.05 mL. When the device is activated, ethanol and water are produced in just a few seconds. The mixing process with the solution was initiated. For each of the five formulations mentioned above, approximately 30% After mixing approximately 2.0 mL of ethanol, add 15 mL of nanoparticle emulsion to Falconchu. The samples were collected in the tube. In each of the five different SNE and CLA-SNE formulations mentioned above, the new A NanoAssemblr (trademark) mixed cartridge was used. The ethanol concentration was Each formulation is reduced by overnight dialysis. After dialysis, the sample is used for analytical characterization. Everything was stored at 4°C.

[0370] Analytical characterization Cationic lipids CLA and squalene are present in MNS as shown in Figure 15A. Therefore, it was incorporated equally into the manufactured squalene CLA-SNE nanoparticles. For all formulation samples described in the example, to remove process ethanol The CLA / squalene (w / w)% ratio (i.e., y-axis) after dialysis in an ethanol solution This was compared to the CLA / squalene (w / w)% (i.e., x-axis) before self-assembly. And the "measured" CLA / squalene (w / w)% after dialysis is at least 35 (w / Up to w)%, it was equal to the "target" (w / w)%. The "target" CL at 100% before self-assembly. Even at A / squalene (w / w)%, in the nanoparticle emulsion manufactured by MNS Compared to the squalene content, more than 70% of the available CLA is contained within CLA-SNE nanoparticles. It is incorporated into the system. The CLA / squalene (w / w)% ratio is determined by inverse phase ULC-CAD. The measurement was performed. CLA was obtained by the microfluidic nanoemulsion self-assembly (MNS) process. It is clearly incorporated into the CLA-SNE that was manufactured using this technology.

[0371] The strength-weighted Z-mean DLS diameter of CLA-SNE formulations manufactured by the MNS process is determined by Ma Measurements were taken using the Lvern ZetaSizer Ultra after dialysis from each preparation. Dissolve aliquots of CLA-SNE samples in 2.0 mL of 20 mM histidine buffer. The samples were diluted 50-fold or 100-fold in pH 5.8. The mean DLS diameter and standard deviation were measured. For each formulation, the measured post-dialysis CLA / squalene (w / w)% was plotted. This is shown in Figure 15B. For each formulation, three DLS measurements were performed at room temperature. The standard deviation was... The standard deviation bar is shown unless it is smaller than the tappoint image. Manufactured by MNS. The intensity-weighted Z-average DLS diameter of the CLA-SNE is in the range of approximately 150-280 nm. This is similar to CLA-SNE nanoparticles produced by high-pressure homogenization. To generate a CLA-SNE adjuvant system with a diameter of , various MNS processes The parameters (for example, those shown above in this embodiment) were controlled.

[0372] CLA-SNE squalene nanoparticle formulation manufactured by the MNS process, pH 5.5 The measured zeta potential is shown in Figure 15C. The zeta potential is Malvern Ze Measured using taSizer Ultra. Post-dialysis CLA-SNE from each preparation. Prepare aliquots of the sample in 2.0 mL of 20 mM citrate BIS TRIS propane solution. Diluted 50 or 100 times in a buffer (pH 5.5). For each formulation, 3 Zeta potential measurements were performed multiple times, except when the standard deviation was smaller than the data point image. The standard deviation bar is shown. 0(w / w)% CLA (i.e., containing CLA) The zeta potential of the CLA-SNE preparation was approximately -5mV. This is shown in Figure 15C. As shown, the addition of CLA significantly increased the zeta potential of the nanoparticles to approximately +10mV.

[0373] Example 15: Optimization of CLA-SNE production by changing aqueous phase pH The CLA-SNE process produces a reversible cationic CLA with an experimentally measured pKa of 6.4. This includes the use of CLA at pH 5.8 in the SNE manufacturing process and the final matrix. The addition of leads to the protonation of CLA, which affects CLA-SNE particles and the manufacturing process. It works to impart an overall net positive charge to the intermediate. The manufacturing of CLA-SNE is finally This results in a 0.8 / 0.2 μm filtration event, and this process step is critical for filtering. Fowling and low product yields were consistently observed, making the process difficult to implement. However, SNE filtration did not present the same challenges as these filtrations of the same scale and was more efficient. It was a typical nanoemulsion filtration process. Importantly, in the absence of CLA, SNE was It does not exhibit the strong positive charge observed in CLA-SNE. A more efficient CLA-SNE filtration system. To produce uncharged CLA-SNE, an aqueous phase (20 mM L-histidine) is used. A series of experiments were conducted in which the pH was adjusted and then used in the production of CLA-SNE. 5. 20 mM with target pH values ​​of 0, 5.7, 5.8, 6.0, 6.2, 7.0, and 7.7 L-histidine was prepared. Then, CLA with a target concentration of 15 mg / mL was formulated. -Each buffer is used as the aqueous phase in the SNE manufacturing process, as described in Example 3. The CLA-SNE manufacturing process proceeded exactly as planned. Upon completion of the homogenization process, The particle size of the CLA-SNE intermediate is determined by Malvern Panalytical Na Measurements were taken using DLS with noZS, followed by filtering with a 0.8 / 0.2 μm PES filter. Filtration was performed using -. The particle size distribution was measured after filtration by DLS, and [CLA] was calculated. Quantitative analysis was performed using UPLC-CAD. 20 mM L-Hypodium with pH 7.0 or 7.7. In the case of CLA-SNE samples prepared using stidine, the material is filtered. Upon application, complete filter fouling was immediately observed, and the collection of filtered material was not possible. Because it was possible, quantification using DLS or ULC-CAD was not possible. Value 0 This is shown for explanatory purposes. In the pre-filtration (pre-filtration) sample, the aqueous phase buffer DLS observed a tendency for particle size to increase as the pH increased (Figure). 16). This relationship is maintained even in filtered samples, and each CLA-SNE sample is filtered. A moderate decrease in particle size was observed afterward. However, the samples at pH 7.0 and 7.7 showed... They are clear exceptions, and they again immediately show complete filter fouling, material Recovery of the material was impossible. In the filtered sample, as the pH of the aqueous phase increased, the final A decrease in [CLA] in the CLA-SNE material was observed (Figure 17). This relationship is Using the aqueous phase at a lower pH in the production of CLA-SNE improves the process yield in final filtration. This indicates an increase in and a more efficient SLA-CAN manufacturing process.

Claims

1. Streptococcus pneumoniae ae) Contains a polysaccharide-carrier protein conjugate and a stable nanoemulsion (SNE) A pneumococcal conjugate composition, The aforementioned SNE is sorbitan trioleate (SPAN-85), polysorbate-20 ( A combination including PS-20 or polysorbate-80 (PS-80), and squalene. Finished product.

2. The aforementioned SNE is 6 μg / mL to 14 mg / mL for SPAN-85, and 6 μg / mL to 14 mg / mL for SPAN-85, and 6 μg / mL to 14 mg / mL. PS-20 or PS-80 in g / mL, and 60 μg / mL to 34 mg / mL The composition according to claim 1, comprising allene.

3. The composition according to claim 1 or 2, wherein the SNE further comprises a cationic lipid.

4. The cationic lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-1 The composition according to claim 3, wherein the composition is 3,16-diene-1-amine.

5. The claim 3 or 4, comprising 30 μg / mL to 2.4 mg / mL of cationic lipids. composition.

6. The aforementioned Streptococcus pneumoniae Each of the niae polysaccharide-carrier protein conjugates is a specific streptococcus. Contains polysaccharides of the S. pneumoniae serotype, The aforementioned Streptococcus pneumoniae serotype is: a) 4, 6B, 9V, 14, 18C, 19F and 23F; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 2 3F; c) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F , 23F and 33F; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5B, 18C, 19A, 19F, 22F, 23F, and 33F; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and bi35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, de-O-acetylation-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 1 5A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and bi35B; h) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 1 7F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F, and 35B; i) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 3 3F and 35B; j) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 1 7F, 19A, 20A, 22F, 23A, 23B, 24F, 31, 33F, and 35B; k) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 1 7F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, and 35B; l) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33 F and 35B; m) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 1 7F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, and 35B; n) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 1 7F, 19A, 20B, 22F, 23A, 23B, 24F, 31, 33F, and 35B; o) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-oacetylation - 15B, 16F, 17F, 19A, 20B, 22F, 23A, 23B, 24F, 31, 3 3F and 35B; and p) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 1 7F, 19A, 20B, 22F, 23A, 23B, 24F, 31, 33F, and 35B A composition according to any one of claims 1 to 5, selected from the group consisting of the following.

7. The composition according to any one of claims 1 to 6, wherein the carrier protein is CRM197. 。

8. The Streptococcus pneumoniae in the above composition (US Pneumoniae) serotypes are 4, 6B, 9V, 14, 18C, 19F and The composition according to claim 7, comprising 23F.

9. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 1 The composition according to claim 7, comprising 4, 18C, 19A, 19F, and 23F9.

10. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 1 The set according to claim 7, consisting of 4, 18C, 19A, 19F, 22F, 23F and 33F. Finished product.

11. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V , 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F The composition according to claim 7, comprising 33F.

12. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V , 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, The composition according to claim 7, comprising 23B, 23F, 24F, 33F, and 35B.

13. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V , 10A, 11A, 12F, 14, 15A, de-O-acetylation - 15B, 18C, 19A , comprising 19F, 22F, 23B, 23F, 24F, 33F and 35B, as described in claim 7 The composition of the listed material.

14. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V , 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, The composition according to claim 7, comprising 23B, 23F, 24F, 33F, and 35B.

15. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F, 19A, 20, 22F, 23A, 23B, 2 The composition according to claim 7, comprising 4F, 31, 33F, and 35B.

16. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, de-O-acetylation - 15B, 16F, 17F, 19A, 20, 22F, The composition according to claim 7, consisting of 23A, 23B, 24F, 31, 33F and 35B 6 thing.

17. The Streptococcus pneumoniae in the above composition (US Pneumoniae) Serotypes: 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15C, 16F, 17F, 19A, 20, 22F, 23A, 23B, 2 The composition according to claim 7, comprising 4F, 31, 33F, and 35B 7.

18. The composition according to any one of claims 1 to 17, wherein the SNE comprises PS-20.

19. pH 5.1–7.0, 5 mM–40 mM histidine and 25 mM–300 mM N The composition according to any one of claims 1 to 18, further comprising aCl.

20. The claim further includes approximately 20 mM histidine and approximately 75 mM NaCl at a pH of approximately 5.

8. A composition according to any one of items 1 to 18.

21. To administer an immunologically effective amount of the composition according to any one of claims 1 to 20 to a patient. Including methods for treating or preventing pneumococcal disease in patients.

22. A composition according to any one of claims 1 to 20 for the treatment or prevention of pneumococcal disease. Use.

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