Pneumococcal conjugate vaccine formulations

Abstract

The present invention provides pneumococcal conjugate vaccine formulations comprising surfactant systems incorporating polysorbate 20 or a combination of a poloxamer and a polyol.

Description

(19) *EP004656650A2* (11) EP 4 656 650 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: 03.12.2025 Bulletin 2025 / 49 (21) Application number: 25203340.2 (22) Date of filing: 20.02.2018 (51) International Patent Classification (IPC): C07K 14 / 315 (2006.01) (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (30) Priority: 24.02.2017 US 201762463220 P (62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC: 18756694.8 / 3 585 803 (71) Applicant: Merck Sharp & Dohme LLC Rahway, New Jersey 07065‑0907 (US) (72) Inventors: • SMITH, William, J. Pennsylvania, 19486 (US) • GIOVARELLI, Cecilia Pennsylvania, 19486 (US) • NAWROCKI, Denise, K. Pennsylvania, 19486 (US) (74) Representative: Merck Sharp & Dohme LLC 120 Moorgate London EC2M 6UR (GB) Remarks: This application was filed on 19‑09‑2025 as a divisional application to the application mentioned under INID code 62. (54) PNEUMOCOCCAL CONJUGATE VACCINE FORMULATIONS (57) The present invention provides pneumococcal conjugate vaccine formulations comprising surfactant systems incorporating polysorbate 20 or a combination of a poloxamer and a polyol. EP 4 65 6 65 0 A 2 Processed by Luminess, 75001 PARIS (FR) Description FIELD OF INVENTION

[0001] The present invention provides pneumococcal conjugate vaccine formulations comprising surfactant systems incorporating polysorbate 20 or a combination of a poloxamer and a polyol. BACKGROUND OF THE INVENTION

[0002] Streptococcus pneumoniae, one example of an encapsulated bacterium, is a significant cause of serious disease world-wide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated there were 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media and 500,000 cases of pneumococcal pneumonia annually in the United States. See Centers for Disease Control and Prevention,MMWRMorbMortalWklyRep1997,46(RR‑8):1‑13.Furthermore, thecomplicationsof thesediseasescanbe significant with some studies reporting up to 8% mortality and 25% neurologic sequelae with pneumococcal meningitis. See Arditi et al., 1998, Pediatrics 102:1087‑97.

[0003] The multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proved invaluable in preventing pneumococcal disease in adults, particularly, the elderly and those at high-risk. However, infants andyoungchildren respondpoorly tounconjugatedpneumococcal polysaccharides.Bacterial polysaccharidesareT-cell- independent immunogens, eliciting weak or no response in infants. Chemical conjugation of a bacterial polysaccharide immunogen toacarrier protein converts the immune response toaT-cell-dependentone in infants.Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM197 have been described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T-cell-stimulating epitopes in their amino acid sequences.

[0004] The pneumococcal conjugate vaccine, Prevnar●, containing the 7most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19Fand23F) causing invasive pneumococcal disease in young childrenand infants at the time,was first licensed in the United States in February 2000. Following universal use of Prevnar● in the United States, there has been a significant reduction in invasive pneumococcal disease in children due to the serotypes present in Prevnar●. SeeCenters for Disease Control and Prevention, MMWRMorbMortal Wkly Rep 2005, 54(36):893‑7. However, there are limitations in serotype coverage with Prevnar● in certain regions of the world and some evidence of certain emerging serotypes in the UnitedStates (for example, 19Aandothers). SeeO’Brien et al., 2004, AmJEpidemiol 159:634‑44;Whitney et al., 2003,N Engl JMed348:1737‑46;Kyawetal., 2006,NEngl JMed354:1455‑63;Hickset al., 2007, J InfectDis196:1346‑54;Traore et al., 2009, Clin Infect Dis 48:S181-S189.

[0005] Prevnar 13● is a 13-valent pneumococcal polysaccharide-protein conjugate vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. See, e.g., U.S. Patent Application Publication No. US 2006 / 0228380 A1, Prymula et al., 2006, Lancet 367:740‑48 and Kieninger et al., Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate VaccineGiven as a 4-Dose Series in Healthy Infants and Toddlers, presented at the 48th Annual ICAAC / ISDA 46th Annual Meeting, Washington DC, October 25‑28, 2008. See, also, Dagan et al., 1998, Infect Immun. 66: 2093‑2098 and Fattom, 1999, Vaccine 17: 126.

[0006] Chinese Patent Application Publication No. CN 101590224 A describes a 14-valent pneumococcal polysac- charide-protein conjugate vaccine including serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F.

[0007] U.S. Pat. No. 8,192,746 describes a 15-valent pneumococcal polysaccharide-protein conjugate vaccine having serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, all individually conjugated to CRM197 polypeptides.

[0008] Multiple carrier protein systems have also been described. See e.g., U.S. Patent Application Publication Nos. 20100209450, 20100074922, 20090017059, 20090010959 and 20090017072.

[0009] Formulations comprising S. pneumoniae polysaccharide-protein conjugates and surfactants including poly- sorbate 80 (PS‑80) and poloxamer 188 (P188) have been disclosed. See U.S. Pat. No. 8,562,999 and U.S. Patent Application Publication No. US20130273098, respectively. SUMMARY OF THE INVENTION

[0010] Thepresent inventionprovides a formulation comprising (i) oneormorepolysaccharide-protein conjugates; (ii) a pH buffered saline solution having a pH in the range from 5.0 to 7.5; (iii) an aluminum salt; and (iv) a surfactant system selected froma)polysorbate20and (b)apoloxamerhavingamolecularweight in the range from1100Da to17,400Daand a polyol selected from propylene glycol (PG) and polyethylene glycol (PEG) 400.

[0011] In certain embodiments, one or more of the polysaccharide-protein conjugates are made in an aprotic solvent, e.g. dimethylsulfoxide (DMSO). In certain aspects of this embodiment, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% or more of the conjugates (on a total protein basis) are prepared in an aprotic solvent such as 2 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 DMSO. Alternatively, 10‑100%, 24%‑100%, or 24‑80% of the conjugates (on a total protein basis) are prepared in an aprotic solvent, e.g., DMSO. In certain aspects of these embodiments, the surfactant system is polysorbate 20 or a poloxamer / polyol combination as described above.

[0012] In certainembodiments, the surfactant systemcomprisesapoloxamerwhichhasamolecularweight in the range from 1100 Da to 17,400 Da, 7,500 Da to 15,000 Da, or 7,500 Da to 10,000 Da. The poloxamer can be poloxamer 188 or poloxamer 407. In certain aspects, final concentration of the poloxamer is from0.001% to 5%w / v, from 0.025% to 1%w / v. In a specific aspect, the polyol is propylene glycol and is at final concentration from 1% to 20% w / v. In another specific aspect, the polyol is polyethylene glycol 400 and is at final concentration from1% to 20%w / v. In certain embodiments, the surfactant systemcomprisespolysorbate20. In certainaspects, thefinal concentrationof thepolysorbate20 is in the range from 0.001% to 10%w / v, or from 0.025% to 2.5%w / v, or from 0.025% to 0.1%w / v. In certain aspects where the surfactant system comprises PS‑20, the formulation further comprises a polyol selected from propylene glycol and polyethylene glycol. The polyethylene glycol or propylene glycol may be at a final concentration of 6% to 20% w / v. In certain embodiments, the polyethylene glycol is polyethylene glycol 400.

[0013] In certain embodiments, the pHbuffered saline solution canhaveapH in the range from5.0 to 7.0. Thebuffer can beselected from thegroup consistingof phosphate, succinate, L-histidine,MES,MOPS,HEPES, acetateor citrate. In one aspect, the buffer is L-histidine at a final concentration of 5mM to50mM,or succinate at a final concentration of 1mM to10 mM. In a specific aspect, the L-histidine is at a final concentration of 20 mM ± 2 mM. The salt in the pH buffered saline solution can bemagnesium chloride, potassium chloride, sodium chloride or a combination thereof. In one aspect, the pH buffered saline solution is sodium chloride. The saline can be present at a concentration from 20 mM to 170 mM.

[0014] In certain embodiments, the polysaccharide-protein conjugates comprise one or more pneumococcal poly- saccharides conjugated toacarrier protein. In certainaspects, thecarrier protein is selected fromCRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid,E. coliLT (heat-labile enterotoxin),E. coliST (heat-stable enterotoxin), exotoxinA fromPseudomonasaeruginosa, and combinations thereof. In one specific aspect, one ormore of the polysaccharide-protein conjugates are conjugated to CRM197. In certain aspects, oneormoreof the polysaccharideprotein conjugates is preparedusing reductiveamination in the non-aqueous solvent DMSO. In this aspect, polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and23Fcanbepreparedusing reductive amination inDMSO, andpolysaccharide protein conjugates fromserotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F can be prepared using reductive amination in aqueous solution. In certain aspects, each dose is formulated to contain: 4µg / mLor 8µg / mLof each saccharide, except for 6B at 8µg / mLor 16µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein.

[0015] The present invention is also directed to a 15-valent pneumococcal conjugate formulation comprising S. pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a CRM197 polypeptide, 20mM L-histidine, 150mMNaCl, 0.2% (w / v) PS‑20 and 250µg / ml APA. In certain aspects, the formulation is formulated as a dosage formcontaining 4µg / mLor 8µg / mLof each saccharide, except for 6B at 8µg / mLor 16 µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein. In certain aspects, polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F prepared using aqueous conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A-D: SDS-PAGE analysis of DTFB process intermediates under nonreducing conditions. A: Samples shown are: Molecular weight standards (lane 1 from left); eluent product from three replicate multimodal anion exchange chromatography runs (MMAEX1,MMAEX2,MMAEX3, lanes2‑4); pooled eluent product frommultimodal anion exchange chromatography runs (MM AEX Pool, lane 5); diafiltered retentate (UF-DR, lane 6); and final bulk intermediate after 0.2-micron filtration (FBI, lane 7). SDS PAGE: NuPAGE 4‑12% Bis-Tris gel; 5 µg / lane; SYPRO Ruby protein gel stain. B: SDS-PAGE analysis (NuPAGE 4‑12% Bis-Tris gel; lanes 2, 4, 8, 10: 5 µg / lane; lane 6: 2 µg / lane; SYPRO Ruby protein gel stain) of DTFB process intermediates run under reducing conditions. Samples shown are: Mark‑12 standards (lanes 1 and 12); purified CRM197 used to generate DTFB (CRM197, lanes 2 and 10); proteolytically-cleaved CRM197 following trypsin digestion step, loaded onto multimodal cation exchange chromato- graphy resin (MM CEX feed, lane 4); product from multimodal cation exchange chromatography (MM CEX product, lane 6); and final bulk intermediate after 0.2-micron filtration (DTFB-FBI, lane 8). C: Samples shown are: molecular weightmarkers (lane1 from left); initial concentrated retentate followingmultimodal cationexchangechromatography (ICR, lane 2); diafiltered retentate (UF-DR, lane 3); concentrated retentate after diafiltration (UF-OCR, lane 4); membrane flush for product recovery (UF-W, lane 5); final pooled retentate plus flush (UF-FR, lane 6); and final bulk intermediateafter 0.2-micron filtration (FBI, lane6).SDS-PAGE:14%Tris-Glycinegel; 8.3‑8.4µg / lane;GelCodeBlue protein gel stain. D: SDS-PAGE analysis (NuPAGE 4‑12% Bis-Tris gel; SYPRO Ruby protein gel stain) of DTFB 3 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 process intermediates run under reducing conditions. Samples shown are: Mark‑12 standards (lanes 1 and 12); proteolytically-cleaved CRM197 following trypsin digestion step, loaded onto multimodal cation exchange chromato- graphy resin (MMCEX feed, lane 2); purified CRM197 used to generate DTFB (CRM197, lane 3); flow-through during column loading (MM CEX flow-through, lane 4); wash of column after loading (MM CEX wash, lane 5); product collected after step elution from multimodal cation exchange chromatography (MM CEX product, lane 7; 10-fold dilutedMMCEXproduct, lane9); and lateelutingproduct collectedafter stepelution frommultimodal cationexchange chromatography (Late eluting MM CEX product, lane 11). Figure 2:DTFBprotein concentration in 100mMpotassium phosphate (KPi) as a function of pH and sodium chloride (NaCl) concentration. Solutions were held overnight at room temperature and then centrifuged. Supernatants assayed by size exclusion chromatography with UV280 absorbance detection. Figure 3:DTFBprotein concentration in potassiumphosphate (KPi) solutions as a function of polysorbate 20 (PS‑20) concentration. Solutions vortexed for 5minutes at room temperature and then centrifuged. Supernatants assayed by size exclusion chromatography with UV280 absorbance detection. Figure 4: ELISA antibody titers for mice immunized with S. pneumoniae serotype 3 polysaccharide conjugated to either CRM197 or DTFB carrier protein and formulated with aluminum phosphate adjuvant (APA). Mice were immunized with one of two independent serotype 3-CRM197 conjugate lots (lots 1 and 2). Figure 5:Survival curves formice immunizedwithS. pneumoniae serotype 3 capsular polysaccharide conjugated to either CRM197 or DTFB carrier protein and formulated with aluminum phosphate adjuvant (APA). Mice were immunized with one of two independent serotype 3-CRM197 conjugate lots (lots 1 and 2). Formulations of APA or saline only were also included in the study as controls. Following immunization, mice were subsequently intraper- itoneally challenged with Serotype 3 bacteria. Figure 6: Laboratory scale stirring study to evaluate the impact of time and stirring on the particle size distribution of a 15-valent pneumococcal polysaccharide (PnPs) conjugate formulation as measured by static light scattering (SLS). All 15 pneumcoccal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. Conjugates were formulated in 20mM L-histidine, pH 5.8, 150mMNaCl and 0.25mg / mL (w / v Al+3) APA for the stirring study. Figure 7:Laboratory scale stirring study to evaluate of the impact of timeand stirring onparticle size distribution of 15- valent pneumococcal polysaccharide conjugate formulations as measured by SLS. All 15 pneumcoccal polysac- charide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. Conjugates were formulated for the stirring study in 20mML-histidine, pH 5.8, 150mMNaCl and 0.25mg / mL (w / vAl+3) APAwith either 0.08% w / v or 0.24% w / v poloxamer 188 (P188). Figure 8A-B: Laboratory scale simulated shipping and handling study of 15-valent pneumococcal polysaccharide conjugate formulations in syringes. All 15 pneumcoccal polysaccharide serotypes were conjugated to CRM197 using reductive amination in aqueous solution. Conjugateswere formulated in 20mML-histidine, pH5.8, 150mMNaCl and 0.25mg / mL (w / v Al+3) APAwithout P188 orwith 0.2%w / vP188. Particle size distribution asmeasured bySLSprior to andafter 24hoursof horizontal rotation (A)andvisual assessement of syringesafter 24hoursof horizontal rotation (B) are shown. Figure 9:Particle size distributions (expressed as a volume-weighted distribution or D[4,3] as measured by SLS) of two 15-valent pneumococcal polysaccharide conjugate formulations with 20 mM L-histidine, pH 5.8, 150 mM NaCl, 0.25 mg / mL (w / v Al+3) APA, and 0.2% w / v P188. Formulation PCV15Aq was comprised of pneumcoccal polysac- charide-CRM197 conjugates, generated by reductive amination in aqueous solution. Formulation PCV15Aq / Non- Aq / ST3-DTFB used a combination of 15 pneumococcal polysaccharide conjugates, some generated by reductive amination in aqueous solution and others by reductive amination in non-aqueous solution; all pneumcoccal poly- saccharide serotypes inPCV15Aq / Non-Aq / ST3-DTFBwere conjugated toCRM197except for serotype3 (ST3),whichwas conjugated to DTFB. Formulations were filled into syringes and horizontally agitated for up to 24 hours at 4°C prior to SLS evaluation. Figures 10A-B: D[4,3] values as measured by SLS of PCV15Aq / Non-Aq / ST3-DTFB formulations containing P188 (A), PS‑80 (B) and PS‑20 (A, B) after stirring and up to 24 hours of horizontal rotation in 1.5 mL HyPak syringes. Figure11:Pneumococcal serotype3-specific IgGconcentrations from InfantRhesusMonkeys (IRM) immunizedwith 15-valent pneumococcal polysaccharide conjugate formulations with aluminum phosphate adjuvant (APA). All formulations contained S. pneumoniae serotype 3 capsular polysaccharide (ST3) conjugated to either CRM197 or DTFB. Formulation PCV15Aq / Non-Aq used a combination of 15 pneumococcal polysaccharide-CRM197 conjugates, some generated by reductive amination in aqueous solution and others by reductive amination in non-aqueous solution. PCV15 formulations contained 0.2% w / v P188 or 0.1% w / v PS‑20 as noted in the figure. Figure 12: Serotype 3 OPA (OPK) titers for Infant Rhesus Monkeys immunized with two 15-valent pneumococcal polysaccharide conjugate vaccines formulated with aluminum phosphate adjuvant (APA) and 0.2% w / v P188. The formulations contained S. pneumoniae serotype 3 capsular polysaccharide (ST3) conjugated to either CRM197 (PCV15Aq) or DTFB (PCV15Aq / Non-Aq / ST3-DTFB). 4 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 Figure 13: Particle size distribution as measured by SLS of PCV15Aq / Non-Aq / ST3-DTFB formulations after 1 hour of stirring and upto 24 hours of horizontal rotation. Formulations contained 0.2% w / v poloxamer 188 and various concentrations of propylene glycol (PG) or polyethylene glycol 400 (PEG400) as noted in the figure. Figures 14A-B: Immunogenicity comparison study in Infant RhesusMonkeys for PCV15Aq / Non-AQ / ST3-DTFB formula- tions containing either 0.2%w / v P188with 15%w / vPGor 0.1%w / v PS‑20 as described in Example 12. Eight animals per group receivedan intramuscular injectionwith either of the two formulationsatT=0 (Dose1), 1month (Dose2) and 2 months (Dose 3) of age. Serum was collected prior to Dose 1 and 2 weeks post dose 1, 2, and 3. The serotype- specific IgG concentrations (IgG GMC) from the pre-immune, post dose‑1, post dose‑2, and post dose 3 serum samples weremeasured as described in Example 10. Panel A shows immunogenicity results for serotypes 1, 3, 4, 5, 6A, 6B, 7F and 9V. Panel B shows immunogenicity results for serotype 14, 18C, 19A, 19F, 22F, 23F, and 33F. Figures 15A-E:D[4,3] values asmeasured by SLS of PCVAq / Non-Aq formulationsas described in Example 13, having different percentages of serotypes made in DMSO (panel A: 24%; panel B: 50%; panel C: 62%; panel D: 79%; and panel E: 100%) containing 0.05%w / v PS‑80, 0.05% w / v PS‑20 and 0.2% w / v PS‑20 after stirring and up to 24 hrs of horizontal rotation in 1.5 mL HyPak Syringes. Figure 16: Immunogenicity results in New ZealandWhite Rabbits for a 15-valent pneumococcal conjugate formula- tion in 20mMHistidine pH 5.8, 150mMNaCl, 250 µg / mL APA, 0.2%w / v PS‑20 having S. pneumoniae polysacchar- ides from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugated to CRM197 using reductive amination in DMSO and S. pneumoniae polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F conjugated to CRM197 using reductive amination in aqueous solution formulated as a dosage form containing 4µg / mL of each saccharide, except for 6B at 8 µg / mL; and about 64 µg / mL CRM197 carrier protein. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention is based, in part, on the discovery that the specific surfactant in multivalent conjugate vaccine formulations can impact the stability of the conjugates and their tendency to aggregate, particularly when one or moreof theconjugatesaremade inanaprotic solvent suchasDMSO.Additionally, somesurfactants require theadditionof a polyol to obtain the requisite stability.

[0018] As used herein, a "protic solvent" is a solvent that has a hydrogen atom bound to an oxygen (as in a hydroxyl group) or a nitrogen (as in an aminegroup). In general terms, any solvent that contains a labileH+ is called a protic solvent.

[0019] As used herein, an "aprotic solvent" refers to a polar aprotic solvent. Such solvents lack an acidic hydrogen and cannot donate hydrogen. Examples of polar aprotic solvents include, but are not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). A non-aqueous solution or solvent is used inter- changeablywithaprotic solvent. Theaprotic solventmayhavesomewaterpresent, for example, up to1%,2%,5%,10%or 20%.

[0020] Asusedherein, the term"polysaccharide" (Ps) ismeant to includeanyantigenicsaccharideelement (or antigenic unit) commonly used in the immunologic and bacterial vaccine arts, including, but not limited to, a "saccharide", an "oligosaccharide", a "polysaccharide", a "liposaccharide", a "lipo-oligosaccharide (LOS)", a "lipopolysaccharide (LPS)", a "glycosylate", a "glycoconjugate" and the like.

[0021] Asused herein, the term "comprises"when usedwith the immunogenic composition of the invention refers to the inclusion of any other components (subject to limitations of "consisting of" language for the antigen mixture), such as adjuvants and excipients. The term "consisting of" when used with the multivalent polysaccharide-protein conjugate mixture refers to a mixture having those particular S. pneumoniae polysaccharide protein conjugates and no other S. pneumoniae polysaccharide protein conjugates from a different serotype.

[0022] As defined herein, the terms "precipitation", "precipitate", "particulate formation", "clouding", and "aggregation" may be used interchangeably and are meant to refer to any physical interaction or chemical reaction which results in the agglomeration of a polysaccharide-protein conjugate. The process of aggregation (e.g., protein aggregation) may be induced by numerous physicochemical stresses, including heat, pressure, pH, agitation, shear forces, freeze-thawing, dehydration, heavy metals, phenolic compounds, silicon oil, denaturants and the like.

[0023] As defined herein, a "surfactant" of the present invention is any molecule or compound that lowers the surface tension of an immunogenic composition formulation. A "surfactant system" comprises a surfactant but may allow for the inclusion of additional excipients such as polyols that increase the effects of the surfactant.

[0024] An immunogenic composition of the inventionmaybeamultivalent composition containing oneormoreantigens conjugated to one or more carrier proteins. In certain embodiments of the invention, the antigen is a saccharide from an encapsulated bacteria. In such vaccines, the saccharides are composed of long chains of sugarmolecules that resemble the surface of certain types of bacteria. Encapsulated bacteria include, but are not limited to,Streptococcus pneumoniae, Neisseriameningitides andHaemophilus influenzae type b. The antigensmay be from the sameorganismormay be from different organisms. In other embodiments of the invention, the antigens are Streptococcus pneumoniae capsular polysaccharides. 5 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55

[0025] In embodiments where two carrier proteins are used, each capsular polysaccharide not conjugated to the first carrier protein is conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule being conjugated to a single carrier protein). In another embodiment, the capsular polysaccharides not conjugated to the first carrier protein are conjugated to twoormore carrier proteins (each capsular polysaccharidemolecule being conjugated to a single carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.

[0026] Diphtheria Toxin, an exotoxin secreted byCorynebacterium diphtheriae, is a classic A-B toxin composed of two subunits (fragments) linked by disulfide bridges and having three domains. Fragment A (DTFA) contains the ADP-ribose catalyticCdomain,whileFragmentB (DTFB) contains the central translocationTdomain andacarboxy terminal receptor- binding R domain. DTFB is the non-toxic moiety constituting approximately 60% of the total amino acid sequence of DT. See e.g., Gill, D. M. and Dinius, L. L., J. Biol. Chem., 246, 1485‑1491 (1971), Gill, D. M. and Pappenheimer, Jr., A. M., J. Biol. Chem., 246, 1492‑1495 (1971), Collier, R. J. and Kandel, J., J. Biol. Chem., 246, 1496‑1503 (1971); and Drazin, R., Kandel, J., and Collier, R. J., J. Biol. Chem., 246, 1504‑1510 (1971).

[0027] The completed amino acid sequence of Diphtheria Toxin has been published. See Greenfield, L., Bjorn, M.J., Horn, G., Fong, D., Buck, G.A., Collier, R.J. and Kaplan, D.A., Proc. Natl. Acad. Sci. USA 80, 6853‑6857 (1983). Specifically, DTFB comprises amino acid residues 194 to 535 of DT.

[0028] TheCRM197 carrier protein is amutant formofDT that is renderednon-toxic by a single aminoacid substitution in Fragment Aat residue 52.CRM197 andDTshare complete sequencehomology in FragmentB.Major T-cell epitopeswere found predominantly in the B fragment of the DT amino acid sequence. See Bixler et al., Adv Exp Med Biol. (1989) 251:175‑80; Raju et al., Eur. J. Immunol. (1995) 25: 3207‑3214; Diethelm-Okita et al., J Infect Dis (2000) 181:1001‑9; and McCool et al., Infect. and Immun. 67 (Sept. 1999), p. 4862‑4869.

[0029] UseofDTFBasdescribedherein includesdiphtheria toxin deletions of theADP-ribosylation activity domain.Use of DTFB also includes variants having at least 90%, 95% or 99% sequence identity including deletions, substitutions and additions. An example of a variant is a deletion ormutation of theCysteine 201. DTFB (C8)means diphtheria toxin deleted of theADP-ribosylatinactivationdomain, andwith cysteine201 removedormutated.UseofDTFBalso includes fragments that cover sequence 265‑450 of DT, which includes the published T-cell epitopes (See Bixler et al., Adv Exp Med Biol. (1989) 251: 175‑80; Raju et al., Eur. J. Immunol. (1995) 25: 3207‑3214). DTFB also includes states ofmonomer, dimer, or oligomers. Use of DTFB also includes any protein complex (excluding full length DT or CRM197), hybrid proteins, or conjugated proteins that contain the DTFB or fragments. Use of DTFB also includes chemically modified DTFB or fragments (i.e. pegylation, unnatural amino acid modification).

[0030] In certain embodiments, the DTFB is produced from enzymatic digestion and reduction of the native DTor the mutant CRM197 with subsequent purification by adsorptive chromatography. Thus, it is envisioned that a purified DTFB, withorwithout amutationat theDTC201 residue, couldbepreparedsimilarly from full lengthnativeorC201-mutatedDTor CRM197, or from variants thereof in which the A-fragment is truncated. It is specifically known that multimodal resins marketed as Capto™Adhere and Capto™MMC and Tris concentrations in excess of 50 mM during the chromatography cycle provide exceptional modes of purifying the cleaved native DTFB.

[0031] In certain embodiments, the preparation of DTFB includes up to 10mMDTT. DTT prevents dimerization caused by disulfide bond formation betweenDTFBmonomers due to free cysteine at residue position 201. In such cases, nickel is not added to the conjugation reaction mixture. However, the conjugation reaction proceeds by the same method otherwise. If DTT is not used, dimerized DTFB may be conjugated to Ps in the presence of nickel to improve the extent of conjugation by sequestering residual, inhibitory cyanide.

[0032] The removal of free cysteine (mutation of the DT C201) in the DTFB is expected to give similar behavior in the multimodal resins. Removal of the free cysteine is expected to eliminate the need for DTTsince dimerization by disulfide bond formation between free cysteine would not be feasible. Increasing the Tris buffer concentration and the sodium chlorideelutionbuffer concentrationhasbeendemonstrated to improve the recoveryofDTFBprotein from theCaptoMMC chromatography resin. It is expected that the DTFB purification can be achieved using other multimodal resins.

[0033] In certain embodiments, the DTFB is expressed recombinantly with or without the mutation of the DT C201 residue and subsequently purified by various techniques known to those skilled in the art.

[0034] In a particular embodiment of the present invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic variant (i.e., toxoid) of diphtheria toxin. In oneembodiment, it is isolated fromcultures ofCorynebacteriumdiphtheria strain C7 (β197) grown in casamino acids and yeast extract-based medium. In another embodiment, CRM197 is prepared recombinantly in accordancewith themethods described inU.S. Pat. No. 5,614,382. Typically, CRM197 is purified through a combination of ultra-filtration, ammonium sulfate precipitation, and ionexchange chromatography. In some embodi- ments,CRM197 is prepared inPseudomonasfluorescensusingPfenexExpressionTechnology™ (Pfenex Inc., SanDiego, CA).

[0035] DTFB and variants thereof can be used as a carrier protein for antigens, including protein (peptides) and saccharides. Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (Diphtheria toxoid), TT (tetanus toxid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent 6 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 Application Publication No. WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; See International Application Patent Publication No. WO 02 / 091998), pneumo- coccal adhesin protein (PsaA),C5apeptidase fromGroupAorGroupBstreptococcus, orHaemophilus influenzaeprotein D, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706‑13) including ply detoxified in some fashion for example dPLY-GMBS (See International Patent Application Publication No. WO 04 / 081515) or dPLY-formol, PhtX, includingPhtA, PhtB, PhtD, PhtEand fusions of Pht proteins for examplePhtDE fusions, PhtBE fusions (See International Patent Application Publication Nos. WO 01 / 98334 and WO 03 / 54007), can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (fromN.meningitidis), PD (Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (See European Patent Nos. EP0378881 and EP0427347), heat shock proteins (See International Patent Application Publication Nos. WO 93 / 17712 and WO 94 / 03208), pertussis proteins (See International Patent Application Publication No. WO 98 / 58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (See International Patent Application Publication No. WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (See Falugi et al., 2001, Eur J Immunol 31:3816‑3824) such as N19 protein (See Baraldoi et al., 2004, Infect Immun 72:4884‑7), iron uptake proteins (See International Patent Application Publication No.WO01 / 72337), toxin A or B ofC.difficile (See InternationalPatentPublicationNo.WO00 / 61761), andflagellin (SeeBen-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.

[0036] Other DT mutants can be used as the second carrier protein, such as CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838‑3844); CRM9, CRM45, CRM102, CRM103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu‑148 to Asp,Gln or Ser and / or Ala 158 toGly and othermutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed inU.S.Pat.No. 5,917,017orU.S.Pat.No. 6,455,673; or fragment disclosed inU.S.Pat.No. 5,843,711.SuchDT mutants can also be used to make DTFB variants where the variants comprise the B fragment containing the epitiope regions.

[0037] In one embodiment, the present invention provides an immunogenic composition comprising polysaccharide- protein conjugates comprising capsular polysaccharides fromat least oneof serotypes1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C,16F, 17F, 18C,19A, 19F, 20, 21, 22A, 22F, 23A,23B, 23F, 24F, 27, 28A,31, 33F, 34, 35A, 35B, 35F, and 38 of Streptococcus pneumoniae conjugated to one or more carrier proteins, and a pharmaceutically acceptable carrier. In certain embodiments of the invention, the immunogenic composition comprises, consists essentially of, or consistsof capsular polysaccharides from2,3, 4, 5, 6, 7, 8, 9, 10,11, 12,13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 serotypes individually conjugated to CRM197. In certain aspects of the invention, CRM197 is the only carrier protein used.

[0038] In certain embodiments, the immunogenic compositions described above optionally further comprise capsular polysaccharides fromoneadditionalS. pneumoniae serotype selected fromat least one of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C,7F,8, 9N,9V,10A,11A, 12F,14,15A,15B,15C,16F,17F,18C,19A,19F,20,21, 22A,22F,23A,23B,23F,24F,27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 conjugated to a second carrier protein (which is distinct in at least one amino acid from the first carrier protein). Preferably, saccharides from a particular serotype are not conjugated to more than one carrier protein.

[0039] In certain embodiments of the invention, the immunogenic composition of the invention further comprises capsular polysaccharides from at least one additional serotype conjugated to a second carrier protein. In these embodiments, the immunogenic composition comprises, consists essentially of, or consists capsular polysaccharides from1, 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, 36, 37, 38, 39, 40, 41, 42, 43, or 44 serotypes individually conjugated to a second carrier protein which is not CRM197.

[0040] In certain embodiments of the invention, the immugenic composition comprises, consists essentially of, or consists of, capsular polysaccharides fromNserotypeswhereN is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44; and capsular polysaccharides from each of the N serotypes are conjugated to the first protein carrier which is CRM197. In other embodiments of the invention, capsular polysaccharides from1, 2, 3 ... or N‑1 serotypes are conjugated to the first protein carrier, and capsular polysaccharides fromN‑1,N‑2,N‑3 ... 1 serotypes are conjugated to the secondprotein carrierwhich is different from CRM197.

[0041] In one specific embodiment of the invention, the present invention provides a 15-valent immunogenic composi- tion comprising, consisting essentially of, or consisting of capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F conjugated to CRM197.

[0042] Capsular polysaccharides from Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and may be sized to some degree by 7 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 known methods (see, e.g., European Patent Nos. EP497524 and EP497525); and preferably by microfluidisation accomplished using a homogenizer or by chemical hydrolysis. In one embodiment,S. pneumoniae strains corresponding to each polysaccharide serotype are grown in a soy-based medium. The individual polysaccharides are then purified through standard steps including centrifugation, precipitation, and ultra-filtration. See, e.g., U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Pat. No. 5,847,112. Polysaccharides can be sized in order to reduce viscosity and / or to improve filterability of subsequent conjugated products. In the present invention, capsular polysaccharides are prepared from one or more of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38.

[0043] The purified polysaccharides are chemically activated to introduce functionalities capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugates may be prepared by known coupling techniques.

[0044] In one embodiment, the chemical activation of the polysaccharides and subsequent conjugation to the carrier protein are achieved by means described in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506. Briefly, the pneumo- coccal polysaccharide is reacted with a periodate-based oxidizing agent such as sodium periodate, potassium periodate, or periodic acid resulting in random oxidative cleavage of vicinal hydroxyl groups to generate reactive aldehyde groups.

[0045] Direct aminative coupling of the oxidized polysaccharide to primary amine groups on the protein carrier (mainly lysine residues) canbeaccomplishedby reductiveamination.Forexample, conjugation is carriedoutby reactingamixture of the activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride in the presence of nickel. The conjugation reaction may be carried out in aqueous solution or in an organic solvent such as DMSO. See, e.g., US2015 / 0231270 A1, EP 0471177 B1, and US2011 / 0195086 A1. At the conclusion of the conjugation reaction, unreacted aldehydes are capped by addition of a strong reducing agent, such as sodium borohydride.

[0046] In one embodiment, prior to formulation, each pneumococcal capsular polysaccharide antigen is individually purified from S. pneumoniae, activated to form reactive aldehydes, and then covalently conjugated using reductive amination with sodium cyanoboroydride in the presence of nickel to the first or second carrier protein. Nickel complexes with residual, inhibitory cyanide from sodium cyanoborohydride reducing agent used for reductive amination.

[0047] In certain embodiments, the conjugation reaction is performed by reductive amination wherein nickel is used for greater conjugation reaction efficiency and to aid in free cyanide removal. Transition metals are known form stable complexes with cyanide and are known to improve reductive methylation of protein amino groups and formaldehyde with sodium cyanoborohydride (Gidley et al., 1982, Biochem J. 203: 331‑334; Jentoft et al., 1980, Anal Biochem. 106: 186‑190).Bycomplexing residual, inhibitory cyanide, theadditionofnickel increases theconsumptionofproteinduring the conjugation of and leads to formation of larger, potentially more immungenic conjugates.

[0048] Variability in free cyanide levels in commercial sodium cyanoborohydride reagent lots may lead to inconsistent conjugation performance, resulting in variable conjugate attributes, including molecular mass and polysaccharide-to- protein ratio. The addition of nickel to the conjugation reaction reduces the level of free cyanide and thus improves the degree of lotto-lot conjugate consistency.

[0049] In another embodiment, the conjugation method may employ activation of polysaccharide with 1-cyano‑4- dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may be coupled directly to an amino group on the carrier protein.

[0050] In another embodiment, a reactive homobifunctional or heterobifunctional group may be introduced on the activatedpolysaccharideby reacting the cyanateesterwith anyof several availablemodalities. For example, cystamineor cysteaminemaybeused topreparea thiolatedpolysaccharidewhich could becoupled to the carrier via a thioether linkage obtained after reaction with a maleimide-activated carrier protein (for example using GMBS) or a haloacetylated carrier protein (for exampleusing iodoacetimide [e.g. ethyl iodoacetimideHCl] orN-succinimidyl bromoacetateorSIAB,orSIA, or SBAP). Inanother embodiment, thecyanateester is reactedwithhexanediamineoradipic aciddihydrazide (ADH)and the resultant amino-derivatised saccharide is conjugated to a free carboxy group on the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistry. Such conjugates are described in International Patent Application Publication Nos. WO 93 / 15760, WO 95 / 08348 and WO 96 / 29094; and Chu et al., 1983, Infect. Immunity 40:245‑256.

[0051] Other suitable conjugation methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide,S--NHS,EDC,andTSTU.Manyaredescribed in InternationalPatentApplicationPublication No.WO98 / 42721. Conjugationmay involve a carbonyl linker whichmay be formed by reaction of a free hydroxyl group of the saccharide with CDI (See Bethell et al., 1979, J. Biol. Chem. 254:2572‑4; Hearn et al., 1981, J. Chromatogr. 218:509‑18) followed by reaction with carrier protein to form a carbamate linkage. This chemistry consists of reduction of the anomeric terminus of a carbohydrate to form a primary hydroxyl group followed by reaction of the primary hydroxyl with CDI to form a carbamate intermediate and subsequent coupling to protein carrier amino groups. The reaction may require optional protection / deprotection of other primary hydroxyl groups on the saccharide.

[0052] Following conjugation, the polysaccharide-protein conjugates are purified to remove excess conjugation reagents as well as residual free protein and free polysaccharide by one or more of any techniques well known to the skilled artisan, including concentration / diafiltration operations, ultrafiltration, precipitation / elution, column chromatogra- 8 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 phy, and depth filtration. See, e.g., U.S. Pat. No. 6,146,902.

[0053] After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composi- tion of the present invention. These pneumococcal conjugates are prepared by separate processes and bulk formulated into a single dosage formulation. Pharmaceutical / Vaccine Compositions

[0054] The present invention further provides compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising, consisting essentially of, or alternatively, consisting of any of the polysaccharide serotype combinations described above together with a pharmaceutically acceptable carrier and an adjuvant. In one embodiment, the compositions comprise, consist essentially of, or consist of 2 to 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 distinct polysaccharide-protein conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to either the first carrier protein or the second carrier protein, and wherein the capsular polysaccharides from at least one of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C,7F,8, 9N,9V,10A,11A, 12F,14,15A,15B,15C,16F,17F,18C,19A,19F,20,21, 22A,22F,23A,23B,23F,24F,27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 of Streptococcus pneumoniae are conjugated to a first carrier protein selected from CRM197, and optionally having additional S. pneumoniae serotypes selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F, and 38 which are conjugated to a second carrier protein (which is distinct in at least one amino acid from the first carrier protein) together with a pharmaceutically acceptable carrier and an adjuvant.

[0055] Formulation of the polysaccharide-protein conjugates of the present invention can be accomplished using art- recognized methods. For instance, 15 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

[0056] In another embodiment, the vaccine composition is formulated in L-histidine buffer with sodium chloride.

[0057] As defined herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of an immunogenic composition of the invention. An immune adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, e.g., inducing no or weak antibody titers or cell-mediated immune response, increase antibody titers to the antigen, and / or lowers the dose of the antigen effective to achieve an immune response in the individual. Thus, adjuvants are often given to boost the immune response and are well known to the skilled artisan. Suitable adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (International Patent Application Publication No. WO 90 / 14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic- blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, (c) Ribi™ adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deaylated monopho- sphorylipid A (MPL™) described in U.S. Pat. No. 4,912,094, trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); and (d) a Montanide ISA; (3) saponin adjuvants, such asQuil A or STIMULON™QS‑21 (Antigenics, Framingham,MA) (see, e.g., U.S. Pat. No. 5,057,540)may be used or particles generated therefrom such as ISCOM (immunostimulating complexes formed by the combination of cholesterol, saponin, phospholipid, andamphipathic proteins) and Iscomatrix● (having essentially the same structure as an ISCOM but without the protein); (4) bacterial lipopolysaccharides, synthetic lipid A analogs such as aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa, and which are described in U.S. Pat. No. 6,113,918; one such AGP is 2‑[(R)‑3-tetradecanoyloxytetradecanoylamino]ethyl 2-Deoxy‑4-O-phosphono‑3- O‑[(R)‑3-tetradecanoyloxytetradecanoyl]‑2‑[(R)‑3-‑ tetradecanoyloxytetradecanoylamino]‑b-D-glucopyranoside, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion; (5) synthetic polynucleotides such as oligonucleotides containing CpG motif(s) (U.S. Pat. No. 6,207,646); (6) cytokines, such as interleukins (e.g., IL‑1, IL‑2, IL‑4, IL‑5, IL‑6, IL‑7, IL‑12, IL‑15, IL‑18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7‑1 and B7‑2, etc; and (7) complement, such as a trimer of complement component C3d. 9 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55

[0058] In another embodiment, the adjuvant is amixture of 2, 3, or more of the above adjuvants, e.g.,. SBAS2 (an oil-in- water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

[0059] Muramyl peptides include, but arenot limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), andN- acetyl-normuramyl-L-alanine‑2‑(1’‑2’ dipalmitoyl-sn-glycero‑3-hydroxyphosphoryloxy)‑ethylamine (MTP-PE), etc.

[0060] In certain embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be an alum- precipitated vaccine or an alum-adsorbed vaccine. Aluminum-salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas,W. (1992; Aluminum salts. Research in Immunology 143:489‑493). The aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel●, Superfos, Amphogel●, aluminum (III) hydroxide, aluminum hydroxyphosphate sulfate (Aluminum Phosphate Adjuvant (APA)), amorphous alumina, trihydrated alumina, or trihydroxyaluminum.

[0061] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is manufactured by blending aluminum chloride and sodium phosphate in a 1:1 volumetric ratio to precipitate aluminum hydroxyphosphate. After the blending process, the material is size-reduced with a high-shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against physiological saline and steam sterilized.

[0062] In certain embodiments, a commercially available Al(OH)3 (e.g. Alhydrogel● or Superfos of Denmark / Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins in a ratio of 50 - 200 g protein / mg aluminum hydroxide. Adsorption of protein is dependent, in another embodiment, on the pI (Isoelectric pH) of the protein and the pH of themedium. A proteinwith a lower pI adsorbs to the positively charged aluminum ionmore strongly than a proteinwith a higher pI. Aluminum salts may establish a depot of Ag that is released slowly over a period of 2‑3 weeks, be involved in nonspecific activation ofmacrophages and complement activation, and / or stimulate innate immunemechanism (possibly through stimulation of uric acid). See, e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.

[0063] Monovalent bulk aqueous conjugates are typically blended together anddiluted.Oncediluted, thebatch is sterile filtered. Aluminum phosphate adjuvant is added aseptically to target a final concentration of 4 µg / mL for all serotypes except 6B, which is diluted to target 8µg / mL, and a final aluminum concentration of 250 µg / mL. The adjuvanted, formulated batch will be filled into vials or syringes.

[0064] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular, aCpG-containingoligodeoxynucleotide (CpGODN). Inanother embodiment, theadjuvant is ODN 1826, which may be acquired from Coley Pharmaceutical Group.

[0065] "CpG-containing nucleotide," "CpG-containing oligonucleotide," "CpG oligonucleotide," and similar terms refer to a nucleotide molecule of 6‑50 nucleotides in length that contains an unmethylated CpGmoiety. See, e.g., Wang et al., 2003,Vaccine21:4297. Inanotherembodiment, anyotherart-accepteddefinitionof the terms is intended.CpG-containing oligonucleotides include modified oligonucleotides using any synthetic internucleoside linkages, modified base and / or modified sugar.

[0066] Methods for use of CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162:6284‑93; Verthelyi, 2006, Methods Mol Med. 127:139‑58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89‑110. Administration / Dosage

[0067] Thecompositionsand formulationsof thepresent inventioncanbeused toprotect or treat ahumansusceptible to infection, e.g., a pneumococcal infection, by means of administering the vaccine via a systemic or mucosal route. In one embodiment, the present invention provides a method of inducing an immune response to a S. pneumoniae capsular polysaccharide conjugate, comprising administering to a human an immunologically effective amount of an immunogenic composition of the present invention. In another embodiment, the present invention provides a method of vaccinating a human against a pneumococcal infection, comprising the step of administering to the human an immunogically effective amount of an immunogenic composition of the present invention.

[0068] Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determinedempirically. InfantRhesusMonkeyanimal dataprovided in theExamplesdemonstrates that that the vaccine is immunogenic.

[0069] "Effective amount" of a composition of the invention refers to a dose required to elicit antibodies that significantly reduce the likelihood or severity of infectivitiy of a microbe, e.g., S. pneumoniae, during a subsequent challenge.

[0070] The methods of the invention can be used for the prevention and / or reduction of primary clinical syndromes caused by microbes, e.g., S. pneumoniae, including both invasive infections (meningitis, pneumonia, and bacteremia), and noninvasive infections (acute otitis media, and sinusitis).

[0071] Administration of the compositions of the invention can include one or more of: injection via the intramuscular, 10 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral / alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage).

[0072] The amount of conjugate in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse effects. Such amount can vary depending upon the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose will comprise 0.1 to 100 µg of each polysaccharide, particularly 0.1 to 10 µg, and more particularly 1 to 5 µg. For example, each dose can comprise 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, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 µg.

[0073] Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determined empirically.

[0074] In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700µg,or1, 1.2, 1.5, 2, 3, 5mgormore. In yetanother embodiment, thedoseofaluminumsalt describedabove isperµgof recombinant protein.

[0075] In a particular embodiment of the present invention, the PCV15 vaccine is a sterile liquid formulation of pneumococcal capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F individually conjugated to CRM197. In one aspect, each dose is formulated to contain: 4 µg / mL or 8 µg / mL of each saccharide, except for 6B at 8 µg / mL or 16 µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein. In one aspect, each 0.5 mL dose is formulated to contain: 2 µg of each saccharide, except for 6B at 4 µg; about 32 µg CRM197 carrier protein (e.g., 32 µg ± 5 µg, ± 3 µg, ± 2 µg, or ± 1 µg), 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and L-histidine buffer. The sodium chloride concentration is about 150 mM (e.g., 150mM± 25mM,± 20mM,± 15mM,± 10mM, or± 5mM) and about 20mM (e..g, 20mM± 5mM,± 2.5mM,± 2 mM, ± 1 mM, or ± 0.5 mM) L-histidine buffer.

[0076] According to any of themethods of the present invention and in oneembodiment, the subject is human. In certain embodiments, the human subject is an infant (less than 1 year of age), toddler (approximately 12 to 24months), or young child (approximately 2 to 5 years). In other embodiments, the human subject is an elderly patient (> 65 years). The compositions of this invention are also suitable for use with older children, adolescents and adults (e.g., aged 18 to 45 years or 18 to 65 years).

[0077] In one embodiment of the methods of the present invention, a composition of the present invention is administered as a single inoculation. In another embodiment, the vaccine is administered twice, three times or four times or more, adequately spaced apart. For example, the composition may be administered at 1, 2, 3, 4, 5, or 6 month intervals or any combination thereof. The immunization schedule can follow that designated for pneumococcal vaccines. For example, the routine schedule for infantsand toddlersagainst invasivediseasecausedbyS.pneumoniae is 2, 4, 6and 12‑15 months of age. Thus, in another embodiment, the composition is administered as a 4-dose series at 2, 4, 6, and 12‑15 months of age.

[0078] The compositions of this invention may also include one or more proteins from S. pneumoniae. Examples of S. pneumoniaeproteins suitable for inclusion include those identified in InternationalPatentApplicationPublicationNos.WO 02 / 083855 and WO 02 / 053761. Formulations

[0079] The compositions of the invention can be administered to a subject by one or more methods known to a person skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, intra- nasally, subcutaneously, intra-peritonealy, and formulated accordingly.

[0080] In one embodiment, compositions of the present invention are administered via epidermal injection, intramus- cular injection, intravenous, intra-arterial, subcutaneous injection, or intra-respiratory mucosal injection of a liquid preparation. Liquid formulations for injection include solutions and the like.

[0081] The composition of the invention can be formulated as single dose vials, multidose vials or as pre-filled syringes.

[0082] In another embodiment, compositionsof thepresent inventionareadministeredorally, andare thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.

[0083] Pharmaceutically acceptable carriers for liquid formulations are aqueous or nonaqueous solutions, suspen- sions, emulsions or oils. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspen- 11 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 sions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.

[0084] The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic, when it is administrated.Hence, for storage the pharmaceutical compositionmay preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.

[0085] The isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Examples of ionic isotonic agents include but are not limited to NaCl, CaCl2, KCl and MgCl2. Examples of non-ionic isotonic agents include but are not limited to mannitol, sorbitol and glycerol.

[0086] It is also preferred that at least one pharmaceutically acceptable additive is a buffer. For some purposes, for example, when the pharmaceutical composition ismeant for infusion or injection, it is often desirable that the composition comprises a buffer, which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.

[0087] The buffer may for example be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate and triethanolamine buffer.

[0088] Thebuffermay furthermore forexamplebeselected fromUSPcompatiblebuffers forparenteral use, inparticular, when the pharmaceutical formulation is for parenteral use. For example the buffer may be selected from the group consisting ofmonobasic acids such as acetic, benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic,malic, succinic and tartaric, polybasic acids suchas citric andphosphoric; andbases suchas ammonia, diethanolamine, glycine, triethanolamine, and Tris.

[0089] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer’s dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycols or polyethylene glycol, Polysorbate 80 (PS‑80), Polysorbate 20 (PS‑20), andPoloxamer 188 (P188) are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.

[0090] The formulations of the inventionmay also contain a surfactant. Preferred surfactants include, but are not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially PS‑20 and PS‑80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO / PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy‑1,2-ethanediyl) groups, with octoxynol‑9 (Triton X‑100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA‑630 / NP‑40); phospholipids such as phosphatidylcholine (le- cithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.

[0091] Mixtures of surfactants can be used, e.g. PS‑80 / Span 85mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS‑80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X‑100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and / or an octoxynol.

[0092] Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as PS‑80) 0.01 to 1% w / v, in particular about 0.1%w / v; octyl‑ or nonylphenoxy polyoxyethanols (such as Triton X‑100, or other detergents in the Triton series) 0.001 to 0.1% w / v, in particular 0.005 to 0.02% w / v; polyoxyethylene ethers (such as laureth 9) 0.1 to 20% w / v, preferably 0.1 to 10% w / v and in particular 0.1 to 1% w / v or about 0.5% w / v.

[0093] In certain embodiments, the composition consists essentially of L-histidine (20 mM), saline (150 mM) and 0.2% w / vPS‑20at a pHof 5.8with 250 ug / mLof APA (AluminumPhosphateAdjuvant). PS‑20 can range from0.005 to 0.3%w / v with the presence of PS‑20 in the formulation controlling aggregation during simulatedmanufacture and in shipping using primary packaging. The process consists of combining blend of up to 44 serotypes in L-histidine, sodium chloride, and PS‑20 then combining this blended material with APA and sodium chloride with or without antimicrobial preservatives.

[0094] As demonstrated herein, the choice of surfactant may need to be optimized for different drug products and drug substances. For multivalent vaccines containing 15 or more serotypes, PS‑20 and P188 are preferred. The choice of chemistry used to prepare the conjugate is believed to be a significant factor that influences the stabilization of the formulation. Inparticular, asexemplifiedbelow, pneumococcal polysaccharide-protein conjugatesprepared inaqueousor DMSO solvent and combined in a multivalent composition show significant differences in stability depending on the particular surfactant systems used for formulation. As described, improved stability was observed with polysorbate 20 alone or with poloxamer 188 in combination with a polyol, particularly when one or more polysaccharide-protein conjugates were prepared in an aprotic solvent such as DMSO.

[0095] The present invention is based, in part, on the discovery that the use of polysorbate 20 or a combination of 12 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 poloxamer 188 and a polyol in formulations containing polysaccharide-protein conjugates prepared using reductive amination, some of which are prepared under aqueous conditions and others of which are prepared under DMSO conditions, aids in the control of manufacturing and shipping stress-induced physico-chemical instability of immunogenic compositionsandprovidesunexpectedly superior properties over other surfactants andstabilizers. Theexactmechanism of how a specifc detergent protects a biotherapeutic is poorly understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential exclusion, air / liquid interface competition between biotherapeutic and surface, surface tension, and / or direct association of the detergent with the biotherpeutic to mask hydrophobic patcheswhich serveas seeds for aggregation. Thepresent invention addressesanongoingneed in theart to improve the stability of and inhibit particulate formation (e.g., aggregation, precipitation) of immunogenic compositions comprising polysaccharide-protein conjugates. The formulations of the invention are believed to provide significant advantages in controlling manufacturing, shipping and handling induced aggregation of complex biotherapeutic over previously used surfactants including Poloxomer 188 and Polysorbate 80.

[0096] It is believed that the protein component in the polysaccharide-protein conjugate plays an important role for aggregation. This is demonstrated in different aggregation phenomena of drug products using conjugates of the same serotype composition but different conjugation solvents. The aprotic solvent used in preparation of a polysaccharide conjugate alters the structure of the protein and may show a different tendency to aggregate in the presence of APA adjuvant. When two or more carrier proteins are used, the weight percentage can be calculated.

[0097] Thus, in certain embodiments of invention, the present invention provides a formulation comprising (i) one or more polysaccharide-protein conjugates; (ii) a pH buffered saline solution having a pH in the range from 5.0 to 7.5; (ii) an aluminum salt; and (iv) a surfactant system selected from (a) polysorbate 20 and (b) a poloxamer having a molecular weight in the range from1100Da to 17,400Da and a polyol selected frompropylene glycol and polyethylene glycol 400. In certain aspects of this embodiment, one or more polysaccharide-protein conjugates are prepared in an aprotic solvent such as DMSO. A range of approximately 10‑100%, 24%‑100%, or 24‑80% of the total mass of protein, can be prepared and conjugated in an aprotic solvent, such as DMSO.

[0098] In certain embodiments, the surfactant system comprises polysorbate 20 (IUPAC name: Polyoxyethylene (20) sorbitan monolaurate; PS‑20) is a commercially available surfactant, commonly referred to as the Tween● 20. In certain embodiments, the final concentration of the polysorbate 20 in the formulations of the invention is in the range from0.001% to 10%w / v, from 0.025% to 2.5%w / v, or 0.025% to 0.3%w / v. A surfactant system comprising polysorbate 20may further comprise a polyol. The polyol may be selected from propylene glycol and polyethylene glycol. In certain aspects, the polyethylene glycol or propylene glycol is at a final concentration of 6% to 20% w / v. In certain aspects, the polyethylene glycol is polyethylene glycol 400.

[0099] In certain embodiments, the surfactant system comprises a poloxamer having a molecular weight in the range from 1100 Da to 17,400 Da and a polyol selected from propylene glycol and polyethylene glycol 400.

[0100] A poloxamer is a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by the tradename Pluronic●. Because the lengths of the polymer blocks can be customized, many different poloxamersexist that haveslightly different properties.For thegeneric term"poloxamer", thesecopolymersarecommonly namedwith the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content (e.g., P407 = Poloxamer with a polyoxypropylene molecular mass of 4,000 g / mol and a 70% polyoxyethylene content). For the Pluronic● tradename, coding of these copolymers starts with a letter to define its physical form at room temperature (L = liquid, P= paste, F = flake (solid)) followed by two or three digits. The first digit (two digits in a three-digit number) in the numerical designation,multiplied by 300, indicates the approximatemolecular weight of the hydrophobe; and the last digit x 10 gives the percentage polyoxyethylene content (e.g., L61 = Pluronic● with a polyoxypropylene molecular mass of 1,800 g / mol and a 10% polyoxyethylene content). See U.S. Pat. No. 3,740,421.

[0101] Examples of poloxamers have the general formula: HO(C2H4O)a(C3H6O)3(C2H4O)aH wherein a and b blocks have the following values: Pluronic● Poloxamer a b Molecular Weight L31 2 16 1100 (average) L35 1900 (average) L44NF 124 12 20 2090 to 2360 L64 2900 (average) L81 2800 (average) 13 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) Pluronic● Poloxamer a b Molecular Weight L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 27 7680 to 9510 F87NF 237 64 37 6840 to 8830 F108NF 338 141 44 12700 to 17400 F127NF 407 101 56 9840 to 14600 Molecular weight units, as used herein, are in Dalton (Da) or g / mol.

[0102] For the formulations, a poloxamer generally hasamolecularweight in the range from1100Da to 17,400Da, from 7,500Da to 15,000Da, or from 7,500Da to 10,000Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407. The final concentration of the poloxamer in the formulations of the invention is from 0.001 to 5% w / v, or 0.025 to 1% w / v.Asurfactant systemcomprisingapoloxamermust further compriseapolyol. In certain aspects, thepolyol is propylene glycol and is at final concentration from1 to 20%w / v. In certain aspects, the polyol is polyethylene glycol 400 and is at final concentration from 1 to 20% w / v.

[0103] Suitable polyols for the formulations are polymeric polyols, particularly polyether diols including, but are not limited to, propylene glycol and polyethylene glycol, Polyethylene glycolmonomethyl ethers. Propylene glycol is available in a range of molecular weights of the monomer from ~425 to ~2700. Polyethylene glycol and Polyethylene glycol monomethyl ether is alsoavailable in a rangeofmolecularweights ranging from~200 to~35000 includingbut not limited to PEG200,PEG300,PEG400, PEG1000PEGMME550,PEGMME600,PEGMME2000,PEGMME3350 andPEGMME 4000.Another polyethyleneglycol is polyethyleneglycol 400.Thefinal concentrationof thepolyol in the formulationsof the invention may be 1 to 20% w / v or 6 to 20% w / v.

[0104] The formulation also contains a pH-buffered saline solution. The buffer may, for example, be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L- histidine, glycine, succinate, HEPES (4‑(2-hydroxyethyl)‑1-piperazineethanesulfonic acid), MOPS (3‑(N-morpholino) propanesulfonic acid), MES (2‑(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. The buffer is capable of buffering a solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In certain aspect of the invention, the buffer selected from the group consisting of phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate or citrate. The buffer may furthermore, for example, be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use. The concentrations of bufferwill range from1mM to50mMor 5mM to50 mM. In certain aspects, the buffer is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM. In certain aspects, the L-histidine is at a final concentration of 20 mM ± 2 mM.

[0105] While the saline solution (i.e., a solution containingNaCl) is preferred, other salts suitable for formulation include but arenot limited to,CaCl2,KCl andMgCl2andcombinations thereof.Non-ionic isotonicagents includingbutnot limited to sucrose, trehalose, mannitol, sorbitol and glycerol may be used in lieu of a salt. Suitable salt ranges include, but not are limited to 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is NaCl, optionally present at a concentration from 20 mM to 170 mM.

[0106] In a preferred embodiment, the formulatons comprise a L-histidine buffer with sodium chloride.

[0107] In certain embodiments of the formulations described herein, the polysaccharide-protein conjugates comprise one or more pneumococcal polysaccharides conjugated to a carrier protein. The carrier protein can be selected from CRM197, diphtheria toxin fragment B (DTFB), DTFBC8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof. In certain aspects, one or more of the polysaccharide-protein conjugates are conjugated to DTFB. In one aspect, all of the polysaccharide-protein conjugates are prepared using aqueous chemisty. As an example, the polysaccharide- protein conjugate formulation can be a 15-valent pneumococcal conjugate (15vPnC) formulation consisting essentially of S.pneumoniaepolysaccharide fromserotypes1,3, 4, 5, 6A, 6B,7F,9V, 14, 18C,19A,19F,22F,23Fand33Fconjugated to a CRM197 polypeptide. In another aspect, one or more of the polysaccharide protein conjugates is prepared using DMSO chemistry. As an example, the polysaccharide-protein conjugate formulation can be a 15-valent pneumococcal conjugate (15vPnC) formulation wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are preparedusingDMSOchemistry andpolysaccharideprotein conjugates fromserotypes1, 3, 4, 5, 9V, 14, 22F, and33Fare prepared using aqueous chemistry.

[0108] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration. In another embodiment, polymeric materials are used; e.g. in microspheres in or an implant.

[0109] The compositions of this invention may also include one or more proteins from S. pneumoniae. Examples of S. 14 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 pneumoniaeproteins suitable for inclusion include those identified in InternationalPatentApplicationPublicationNos.WO 02 / 083855 and WO 02 / 053761.

[0110] Having described various embodiments of the invention with reference to the accompanying description and drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes andmodificationsmaybe effected therein by one skilled in the art without departing from the scopeor spirit of the invention as defined in the appended claims.

[0111] The following examples illustrate, but do not limit the invention. EXAMPLES EXAMPLE 1: Preparation of DTFB Carrier Protein Use of Multimodal Anion Exchange Chromatography for DTFB Preparation

[0112] Purified CRM197, obtained through expression in Pseudomonas fluorescens as previously described (See International Patent Application Publication No. WO 2012 / 173876 A1), was digested with recombinant trypsin using a 1:500molar ratio of trypsin to CRM197 for approximately 1 hour at approximately 22°C in 50mMTris, pH 8.0. Dithiotheritol (DTT) in 50mMTris, pH 8was then added to a final concentration of 5mM for 30minutes at approximately 22°C to reduce the disulfide bond between the A and B fragments of the proteolytically-cleaved CRM197.

[0113] The digestion reaction was then loaded onto a multimodal anion exchange chromatography column (Cap- to™Adhere, GE Healthcare) equilibrated with 50 mM Tris, pH 8. The column was washed with 50 mM Tris, pH 8, and the DTFB product was eluted with a gradient of 0.45 M to 0.65 M sodium chloride in 50 mM Tris, pH 8. The product was concentrated and diafiltered against 10 mM potassium phosphate, pH 8 using a 5 kDa Nominal Molecule Weight Cut-Off (NMWCO) tangential flow ultrafiltration membrane. The retentate containing DTFB product was 0.2-micron-filtered and stored at 2‑8°C. Product concentrationwas determinedby absorbance at 280 nmandpurity was assessed bySDS-PAGE under nonreducing conditions.

[0114] Results show that the multimodal anion exchange chromatography eluent contained relatively pure DTFB, present primarily as amonomer with a small fraction of dimer. See Figure 1A.Dimer formation was attributed to disulfide bond formation between DTFB monomers. As exemplified in the following sections, DTT has been used in the chromatography and ultrafiltration steps to minimize potential for dimer formation. Use of Multimodal Cation Exchange Chromatography for DTFB Preparation

[0115] Due to the presence of DTFB dimers, an alternative purification method was investigated. Purified CRM197, obtained through expression in Pseudomonas fluorescens as described above, was diluted to a protein concentration of approximately 1 mg / mL using 300 mM Tris, pH 7.5. Trypsin was added to the protein solution using a trypsin to CRM197 molar ratio of approximately 1:3250. The solution was incubated for approximately 20 hours at room temperature. DTT in 300 mM Tris, pH 7.5 was then added to a final concentration of 10 mM DTT to reduce the disulfide bond between the proteolytically-cleaved CRM197, separating the A and B fragments.

[0116] After approximately 75 minutes, the reduced protein solution was loaded onto a multimodal cation exchange chromatography column (Capto™MMC,GEHealthcare). The columnwas equilibrated at 2‑8°Cwith 300mMTris, 10mM DTT, pH 7.5 prior to loading the protein solution. After loading, the column was washed at 2‑8°Cwith 300mMTris, pH 7.5 containing 200mMsodium chloride and 10mMDTT, and the product was then eluted at 2‑8°Cwith 1Msodium chloride in 300 mM Tris, pH 8.5. Approximately 0.002% w / v PS‑20 was added to the batch prior to concentration at 2‑8°C using a 5 kDa NMWCO tangential flow ultrafiltration membrane. After concentration, additional PS‑20 was added to the batch to a concentration of 0.02%w / vPS‑20, and thebatchwasdiafiltered against 100mMpotassiumphosphate, 10mMDTT, pH8. The batch was further concentrated using tangential flow filtration with a 5 kDa membrane. The final retentate was 0.2- micron filtered and stored frozen or at 2‑8°C prior to conjugation.

[0117] DTFB samples were analyzed by SDS-PAGE under reducing conditions (Figures 1B and 1C). Densitometric analysis of the gel shows that the final bulk intermediate after 0.2-micron filtration (FBI) has purity of > 98%.

[0118] The final bulk intermediate (FBI) shown in Figure 1C was analyzed by liquid chromatography with mass spectrometry (LC-MS) to measure intact protein mass. LC-MS analysis was performed on samples after reduction with DTT. Deconvolution of the raw data from the main peak resulted in a measured mass of 37,194.2 Da. This mass measurement is consistent with the theoretical mass of 37,194.4 Da for the DTFB, confirming the expected amino acid sequence.

[0119] A peptide map was obtained from a combination of trypsin, endoproteinase Asp-N, and endoproteinase Glu-C digestions. The sample was subjected to reductive alkylation with iodoacetamide in the presence of 6 M guanidine-HCl, andwas digested for approximately 16‑17 hours at 37°Cwith each enzyme separately. The digestions were quenched by 15 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 the addition of formic acid. Peptides were separated and analyzed by LC-MS. Using a combination of the peptides identified by the separate digestions, amino acid sequence coverage was approximately 98%. The peptides found were consistent with the predicted DTFB sequence. Alternative Multimodal Cation Exchange Chromatography Process for DTFB Purification

[0120] Purified CRM197, obtained through expression inPseudomonas fluorescens as described above, was diluted to a protein concentration of approximately 5 mg / mL using 300 mM Tris, pH 7.5. Trypsin was added to the protein solution using a trypsin to CRM197molar ratio of approximately 1:3000. The solution was incubated for approximately 15‑20 hours at approximately 22°C. DTT in 300mMTris, pH 7.5 was then added to a final concentration of ≥ 10mMDTT to reduce the disulfide bond between the proteolytically-cleaved CRM197, separating the A and B fragments.

[0121] The reduced protein solution was loaded at onto a multimodal cation exchange chromatography column (Capto™MMC, GE Healthcare) at approximately 25 g protein per L resin. The column was equilibrated at approximately 22°C with 300 mM Tris, 10 mMDTT, pH 7.5 prior to loading the protein solution. After loading, the column was washed at approximately 22°C with 300 mM Tris, pH 7.5 containing 200 mM sodium chloride and 10 mM DTT, and the product was then eluted at 22°C with 1M sodium chloride in 300mMTris, pH 8.5. DTFB product from themultimodal cation exchange chromatography can be diafiltered and concentrated using a 5 kDaNMWCO tangential flow ultrafiltrationmembrane and 0.2-micron filtered as described above.

[0122] DTFB samples from the multimodal cation exchange process were analyzed by SDS-PAGE under reducing conditions (Figure 1D).Densitometric analysis of the gel shows that theDTFB protein product eluted from themultimodal cation exchange column is highly purified. Effects of Buffer pH, Ionic Strength, and PS‑20 Surfactant Concentration on DTFB Stability during Ultrafiltration

[0123] Buffer pH, ionic strength and PS‑20 surfactant studies were conducted to address protein particle formation observed during development of the DTFB ultrafiltration step. DTFBwas adjusted to 100mMpotassium phosphate at pH 7, 7.5, or pH 8 with 0, 200, or 500 mM sodium chloride. Differential scanning calorimetry was used to assess the stability (melting temperature, Tm) of the DTFB solutions as function of pH and sodium chloride concentration. As shown in Table 1, increasing thepH from7 to8 increased theDTFBTmbyapproximately 3°C.Sodiumchloride in the rangeof 0 to 500mM sodium chloride did not significantly impact Tm at the pH values investigated. Table 1: Stability of DTFB solutions as a function of buffer pH and sodium chloride concentration as measured by differential scanning calorimetry. pH of potassium phosphate buffer Sodium chloride concentration (mM) Melting temperature, Tm (°C) 7 0 50.6 200 50.4 500 50.6 7.5 0 51.5 200 51.3 500 51.2 8 0 52.8 200 53.2 500 53.3

[0124] In a separate study, purified DTFB was adjusted to pH 6.0, pH 7.0, and pH 8.5 in 100 mM potassium phosphate with0, 150, 500,and1000mMsodiumchloride.Solutionswereheldovernight at room temperatureand thencentrifuged to removeprecipitated protein. Supernatantswereassayed for protein concentration by size exclusion chromatographywith UV280absorbancedetection (Figure2).Supernatant protein concentration in this studywasnot affectedby solutionpHat 0 and 150 mM sodium chloride. However, at higher sodium chloride concentrations (500 and 1000 mM), results show a pronounced decrease in supernatant protein concentration, indicative of reduced DTFB stability, as buffer pH decreased from pH 7.0 or 8.5 to 6.0.

[0125] Theeffect ofPS‑20concentrationonDTFBstabilitywas studiedbyadding increasingamounts ofPS‑20 toDTFB solutions in 50 and 100 mM potassium phosphate, pH 8 solutions and in 50 mM potassium phosphate, 150 mM sodium 16 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 chloride, pH 8. Solutions were vortexed for 5 minutes and then centrifuged. Supernatants were assayed for protein concentration by sizeexclusion chromatographywithUV280absorbancedetection (Figure3).Significant vortex-induced protein losswas observed in samples containing noPS‑20.DTFB recoverywas notably improved in samples containing ≥ 0.01% w / v PS‑20. EXAMPLE 2: Preparation of S. pneumoniae Capsular Polysaccharides

[0126] Methodsof culturingpneumococci arewell known in theart. See, e.g.,Chase, 1967,Methodsof Immunologyand Immunochemistry 1:52. Methods of preparing pneumococcal capsular polysaccharides are also well known in the art. See, e.g., European Patent No. EP0497524. Isolates of pneumococcal subtypes are available from the American Type CultureCollection (Manassas, VA). Thebacteria are identifiedasencapsulated, nonmotile,Gram-positive, lancet-shaped diplococci that are alpha-hemolytic on blood-agar. Subtypes can be differentiated on the basis of Quelling reaction using specific antisera. See, e.g., U.S. Pat. No. 5,847,112.

[0127] Cell banks representing each of the S. pneumoniae serotypes of interest were obtained from the Merck Culture Collection (Rahway, NJ) in a frozen vial. A thawed seed culture was transferred to the seed fermentor containing a pre- sterilizedgrowthmedia appropriate forS. pneumoniae.Theculturewasgrown in the seed fermentorwith temperature and pH control. The entire volume of the seed fermentor was transferred to a production fermentor containing pre-sterilized growth media. The production fermentation was the final cell growth stage of the process. Temperature, pH, and the agitation rate were controlled.

[0128] The fermentation process was terminated via the addition of an inactivating agent. After inactivation, the batch was transferred to the inactivation tankwhere it was held at controlled temperature andagitation.Cell debriswas removed using a combination of centrifugation and filtration. The batch was ultrafiltered and diafiltered. The batch was then subjected to solvent-based fractionations that remove impurities and recover polysaccharide. EXAMPLE 3: Conjugation of Polysaccharides to DTFB Carrier Protein using Reductive Amination in Aqueous Solu- tion Preparation of Serotype 3-DTFB (ST3-DTFB) Conjugate for Mouse Immunogenicity Studies

[0129] Purified serotype 3polysaccharide obtained as described inExample 2wasdissolved inwater. Ps size reduction toanaveragemolecularweightof approximately200kDawasperformedusingaprobesonicatorwith thesamplecooled in ice. The sonicated sample was 0.2micron-filtered and stored at 2‑8°C. The polysaccharide solution was concentrated by diafiltration against a 30 kDa NMWCO tangential flow filtration membrane.

[0130] Polysaccharidewasprepared for conjugation using sodiummetaperiodate oxidation (SeeAndersonet al., 1986, J. Immunol. 137:1181‑1186; and U.S. Patent Application Publication No. US20110195086). A 100 mM sodiummetaper- iodate solutionwasadded to thepolysaccharide solution in 50mMsodiumacetate. Thesamplewasmixed for 14‑18hours at 19‑25°C protected from light. Ethylene glycol (100:1 molar excess over polysaccharide repeat units) was added and mixedanadditional 16‑18hoursat 19‑25°C toquench residual sodiummetaperiodate andstop theoxidation reaction. The resulting solutionwas diafiltered against 10 volumes of water. The oxidized polysaccharide solutionwas stored in aliquots at ‑70°C.

[0131] The periodate-oxidized polysaccharide was mixed with DTFB prepared as described in Example 1 (using multimodal anionexchangechromatography) at apolysaccharide to proteinmass ratio of 0.6:1.Potassiumphosphate, pH 6.4 andnickel chloridewereadded to final concentrations of 145mMand2.2mM, respectively. Sodiumcyanoborohydride toapproximately1‑2molarequivalentswas thenadded.The reactionwasprotected from light andcarriedoutoveraperiod of 120 hours at 2‑8°C.

[0132] The mixture was then dialyzed against 2 changes of 25 mM potassium phosphate buffer, pH 6.4, 0.3 M sodium chloride at 2‑8°C for a total of 14 - 18 hours. Insoluble materials were removed by brief centrifugation, and the conjugate was polished by size exclusion chromatography (SEC) to reduce free polysaccharide and protein. The SEC-polished conjugate was concentrated over a 30 kD NMWCO centrifugal concentrator. Preparation of Serotype 3-DTFB Conjugate for Infant Rhesus Monkey Immunogenicity Study

[0133] Purified Serotype 3 pneumococcal capsular polysaccharide powder was dissolved in water and 0.45-micron filtered. The batch was homogenized to reduce the molecular mass of the Ps and 0.22-micron filtered. Size reduction of Streptococcus pneumoniae polysaccharide, prior to conjugation, has been described previously as means to generate polysaccharide with more specific, reproducible, and manageable physical properties (Marburg et al., 1997, US Patent 5,623,057). As described by Marburg et al., polysaccharide size reduction is known to increase solubility and filterability and to reduce polydispersity and viscosity, thereby improving conjugation consistency and ease of conjugation. Poly- 17 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 saccharide size reductionof serotype19Fpolysaccharide fromS.pneumoniaeusinghomogenizationhasbeendescribed previously (Lander et al., 2000, Biotechnol. Prog. 2000, 16, 80‑85). The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0134] 50mMsodiumacetatewas thenadded, andpolysaccharideactivationwas initiatedwith theadditionof a 100mM sodium metaperiodate solution to form reactive aldehydes on the polysaccharide. The batch was incubated at approxi- mately 22°C for approximately 12hours. Thebatchwasdiafilteredagainst 10mMpotassiumphosphate, pH6.4 usinga10 kDa NMWCO tangential flow ultrafiltration membrane at ≤ 8°C and the product-rich retentate was concentrated.

[0135] Activated polysaccharide solution was blended with water and 1.5 M potassium phosphate, pH 7.0. Purified DTFB was 0.2-micron filtered, and then combined with the bufferadjusted polysaccharide solution at a polysaccharide to protein mass ratio of 1.3:1. The solution was then 0.2 micron filtered. Nickel chloride was added to the batch to a final concentration of approximately 2 mM using a 100 mM nickel chloride stock solution. Sodium cyanoborohydride (2 moles permole of polysaccharide repeating unit) was then added. The batchwas allowed to react for approximately 120 hours at approximately 10°C to maximize consumption of polysaccharide and protein.

[0136] Following the conjugation reaction, the batchwas diluted to a polysaccharide concentration of approximately 3.5 g / L, cooled to 2‑8°C, 1.2micron filtered, and diafiltered against 100mMpotassiumphosphate, pH7.0 at 2‑8°Cusing a100 kDa NMWCO tangential flow ultrafiltration membrane. The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide / L and pH-adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1mole permole of polysaccharide repeating unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

[0137] The batchwas then concentrated and diafiltered against 10mML-histidine in 150mMsodium chloride, pH 7.0 at 2‑8°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The retentate was 0.2 micron filtered and adjusted to apolysaccharide concentration of 1.0 g / Lwith additional 10mML-histidine in 150mMsodiumchloride, pH7.0. The batch was dispensed into aliquots and frozen at ≤ ‑60°C. EXAMPLE 4: Conjugation of Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F to CRM197 using Reductive Amination in Aqueous Solution

[0138] The different serotype polysaccharides were individually conjugated to purified CRM197 carrier protein using a common process flow. Polysaccharide was dissolved, size reduced, chemically activated and buffer-exchanged by ultrafiltration. Purified CRM197 was then conjugated to the activated polysaccharide utilizing NiCl2 (2 mM) in the reaction mixture, and the resulting conjugate was purified by ultrafiltration prior to a final 0.2 micron filtration. Several process parameters within each step, such as pH, temperature, concentration, and time were controlled to serotype-specific values in section below. Polysaccharide size reduction and oxidation

[0139] Purified pneumococcal capsular polysaccharide powder was dissolved in water, and all serotypes, except serotype 19A, were 0.45-micron filtered. Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F, 22F, 23F, and 33F were homogenized to reduce the molecular mass of the polysaccharide. Serotype 18C was size-reduced by either homo- genization or acid hydrolysis at ≥ 90°C. Serotype 19A was not size reduced due to its relatively low starting size. Homogenization pressure and number of passes through the homogenizer were controlled to serotype-specific targets (150‑1000 bar; 4‑7 passes) to achieve a serotype-specificmolecularmass. Size-reduced polysaccharidewas 0.2-micron filtered and then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for acid-hydrolyzed serotype 18C.

[0140] The polysaccharide solution was then adjusted to a serotype-specific temperature (4‑22°C) and pH (4‑5) with a sodium acetate buffer to minimize polysaccharide size reduction due to activation. For all serotypes (except serotype 4), polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype-specific, ranging from approximately 0.1 to 0.5 moles of sodium metaper- iodate permole of polysaccharide repeating unit. The serotype-specific charge of sodiummetaperiodatewas to achieve a target level of polysaccharide activation (moles aldehyde permole of polysaccharide repeating unit). For serotype 4, prior to thesodiummetaperiodateaddition, thebatchwas incubatedatapproximately50°CandpH4.1 topartially deketalize the polysaccharide.

[0141] For all serotypes, with the exception of serotypes 5 and 7F, the activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for acid-hydrolyzed serotype 18C. Serotypes 5 and 7F were diafiltered against 10 mM sodium acetate. Ultrafiltration for all serotypes was conducted at 2‑8°C. 18 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 Polysaccharide conjugation to CRM197

[0142] Oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate, pH 6.0 or pH 7.0, depending on the serotype. The buffer pH selected was to improve the stability of activated polysaccharide during the conjugation reaction. Purified CRM197, obtained through expression in Pseudomonas fluorescens as previously de- scribed (See InternationalPatentApplicationPublicationNo.WO2012 / 173876A1),was0.2-micronfilteredandcombined with thebuffered polysaccharide solution at a polysaccharide toCRM197mass ratio ranging from0.4 to 1.0w / wdepending on the serotype. Themass ratiowas selected to control the polysaccharide toCRM197 ratio in the resulting conjugate. The polysaccharide and phosphate concentrations were serotype-specific, ranging from 3.6 to 10.0 g / L and 100 to 150 mM, respectively, depending on the serotype. The serotype-specific polysaccharide concentration was selected to control the sizeof the resultingconjugate.Thesolutionwas then0.2-micronfiltered.Nickel chloridewasadded toapproximately2mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeating unit) was added. Conjugation proceeded for a serotype-specific duration (72 to 120 hours) to maximize consumption of polysaccharide and protein.

[0143] Acid-hydrolyzed serotype 18C was conjugated at 37°C in 100 mM potassium phosphate at approximately pH 8 with sodium cyanoborohydride using polysaccharide and protein concentrations of approximately 12.0 g / L and 6.0 g / L, respectively. Reduction with sodium borohydride

[0144] Following the conjugation reaction, the batchwas diluted to a polysaccharide concentration of approximately 3.5 g / L, cooled to 2‑8°C, and 1.2-micron filtered. All serotypes (except serotype 5) were diafiltered against 100mMpotassium phosphate, pH7.0 at 2‑8°Cusing a100 kDaNMWCO tangential flowultrafiltrationmembrane. Thebatch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide / L and pH-adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodiumborohydride (1mole permole of polysaccharide repeating unit) was added. 1.5Mpotassium phosphate, pH 6.0 was later added. Serotype 5 was diafiltered against 300 mM potassium phosphate using a 100 kDa NMWCO tangential flow ultrafiltration membrane. Final filtration and product storage

[0145] Thebatchwas then concentratedanddiaftilteredagainst 10mML-histidine in 150mMsodiumchloride, pH7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The retentate batch was 0.2 micron filtered.

[0146] Serotype 19F was incubated for approximately 7 days at 22°C, diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 100 kDaNMWCO tangential flow ultrafiltrationmembrane, and 0.2-micron filtered.

[0147] Thebatchwas adjusted to a polysaccharide concentration of 1.0 g / Lwith additional 10mML-histidine in 150mM sodium chloride, pH 7.0. The batch was dispensed into aliquots and frozen at ≤ ‑60°C. EXAMPLE 5: Methods for the Conjugation of Serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F to CRM197 using Reduc- tive Amination in Dimethylsulfoxide

[0148] The different serotype polysaccharides were individually conjugated to purified CRM197 carrier protein using a common process flow. Polysaccharide was dissolved, sized to a target molecular mass, chemically activated and buffer- exchanged by ultrafiltration. Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in dimethylsulfoxide (DMSO). Redissolved polysaccharide andCRM197 solutionswere then combined and conjugated as described below. The resulting conjugatewas purified by ultrafiltration prior to a final 0.2-micron filtration. Several process parameters within each step, such as pH, temperature, concentration, and time were controlled to serotype-specific values in section below. Polysaccharide size reduction and oxidation

[0149] Purified pneumococcal capsular polysaccharide powder was dissolved in water, and all serotypes, except serotype 19A, were 0.45-micron filtered. All serotypes, except serotypes 18C and 19A, were homogenized to reduce the molecular mass of the polysaccharide. Homogenization pressure and number of passes through the homogenizer were controlled to serotype-specific targets (150‑1000 bar; 4‑7 passes). Serotype 18Cwas size-reduced by acid hydrolysis at ≥ 90°C. Serotype 19A was not sized-reduced.

[0150] Size-reducedpolysaccharidewas0.2-micronfilteredand thenconcentratedanddiafilteredagainstwaterusinga 10 kDa NMWCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for serotype 18C.

[0151] The polysaccharide solution was then adjusted to a serotype-specific temperature (4‑22°C) and pH (4‑5) with a 19 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 sodium acetate buffer. Polysaccharide activation was initiated with the addition of a sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype-specific, ranging from approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit.

[0152] For all serotypes, the activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for serotype 18C. Ultrafiltration for all serotypes was conducted at 2‑8°C. Polysaccharide conjugation to CRM197

[0153] Purified CRM197, obtained through expression in Pseudomonas fluorescens as previously described (See International Patent Application Publication No. WO 2012 / 173876 A1), was diafiltered against 2 mM phosphate, pH 7 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and 0.2-micron filtered.

[0154] The oxidized polysaccharide solution was formulated with water and sucrose in preparation for lyophilization. The protein solution was formulated with water, phosphate buffer, and sucrose in preparation for lyophilization. Sucrose concentrations ranged from 1 to 5% to achieve optimal redissolution in DMSO following lyophilization.

[0155] Formulated polysaccharide andCRM197 solutionswere individually lyophilized. Lyophilized polysaccharide and CRM197 materials were redissolved in DMSO and combined using a tee mixer. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added, and conjugation proceeded for a serotype-specific duration (1 to 48 hours) to achieve a targeted conjugate size. Reduction with sodium borohydride

[0156] Sodium borohydride (2 mole per mole of polysaccharide repeating unit) was added following the conjugation reaction. The batchwas diluted into 150mMsodiumchloride at approximately 4°C. Potassiumphosphate bufferwas then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Final filtration and product storage

[0157] Each batchwas then concentrated and diaftiltered against 10mML-histidine in 150mMsodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The retentate batch was 0.2-micron filtered.

[0158] Serotype 19F was incubated for approximately 5 days, diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at approximately 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and 0.2-micron filtered.

[0159] The batch was diluted with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 and dispensed into aliquots and frozen at ≤ ‑60°C. EXAMPLE 6: Mouse Immunogenicity Study using ST3-DTFB Monovalent Conjugate Formulation

[0160] The immungenicity of ST3-DTFB compared to ST3-CRM197 was evaluated in a mouse model. Adjuvanted formulations for administration tomice were prepared bymixing 24µL of sterile-filtered conjugate (1:10 in saline - 0.1258 mgDTFBorCRM197-conjugatedpolysaccharidepermL)with62µLofAPA,and3.664mlof sterile saline for adoseof 0.08 µg of polysaccharide and 5 µg of aluminum per 100 µL. The formulated vaccines were stored in individual borosilicate stoppered vials at 2‑8°C to support individual immunizations.

[0161] ST3-DTFB was evaluated in 6‑8 week old female Balb / C mice (n=10 / group). Mice were immunized with ST3- DTFB / APA and two ST3-CRM197 / APA lots made using unique ST3-DTFB and ST3-CRM197 conjugate preparations prepared as described in Examples 3 and 4. The ST3PnPs concentration was 0.08µg per dose in a 0.1ml volume, with 5 µg of APA, given intraperitoneally on days 0, 14 and 28. Sera were collected prior to study start (pre) and on day 39, post- dose3 (PD3) and tested in aST3WHOELISA [per standardizedWorldHealthOrganization (WHO)protocols] aswell as in a protein carrier ELISA (CRM197 and DTFB). Mice were challenged intraperitoneally withS. pneumoniae serotype 3 (207 CFU / 0.5 ml) on day 49.

[0162] WHO ELISA results (Figure 4) showed mice immunized with both ST3-DTFB / APA and ST3-CRM197 / APA had similar PD3 PnPs 3 titers. Mice immunized with ST3-DTFB / APA and ST3-CRM197 / APA had ≥ 90% protection against a serotype 3 challenge, which was significantly higher relative to the negative control saline and APA immunized mice (Figure 5). 20 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 EXAMPLE 7: Formulation of a 15-valent Pneumococcal Conjugate Vaccine with Different Surfactants and Stabilizers

[0163] Pneumococcal polysaccharide-protein conjugatespreparedasdescribedabovewereused for the formulationof a 15-valent pneumococcal conjugate vaccine (PCV15) having serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. The formulations were prepared using pneumococcal polysaccharide-CRM197 conjugates generated by reductive amination in aqueous solutions (Example 4) or in DMSO (Example 5). ST3-DTFB conjugates were prepared as per Example 3. The required volumes of bulk conjugates needed to obtain the target final concentration of individual serotypewerecalculatedbasedon thebatchvolumeand thebulk polysaccharide concentrations.The15conjugateswere combined with the excipients selected from sodium chloride, L-histidine, pH 5.8 buffer with PS‑20, PS‑80, or P188.

[0164] The sterile formulated bulk was mixed gently during and following its blending with bulk Aluminum Phosphate Adjuvant (APA) with or without propylene glycol (PG) and polyethylene glycol 400 (PEG400). Two concentrations of conjugates and APA were studied in the various formulations. One contained 8 µg / mL serotype 6B polysaccharide, 4 µg / mL polysaccharide for all other serotypes, and 250 µg / mL APA. The other contained 16 µg / mL serotype 6B polysaccharide, 8 µg / mL polysaccharide for all other serotypes, and 500 µg / mL APA. The formulated vaccines were stored at 2 - 8°C. EXAMPLE 8: Impact of Excipients on Stability of a Pneumococcal Conjugate Vaccine Formulation Containing Conju- gates Generated by Reductive Amination in Aqueous Solution

[0165] The stability of a 15-valent Pneumococcal Conjugate Vaccine (PCV15), prepared as described in Example 7, was evaluated for various excipient conditions after stirring, recirculation, and rotational agitation studies to simulate manufacturing and shipping stresses that could occur. PCV15 was prepared with 20 mM L-histidine, pH 5.8, 150 mM sodium chloride, and either one of the two concentrations of conjugates and APA listed in Example 7. Since the results were very similar between the two formulations at different concentrations of conjugates and APA, only results for PCV15 formulation containing lower concentrations of conjugates and APAwere shown in this Example. For the stirring studies, the PCV15 formulations were mixed using a magenetic stir bar in a glass vessel. For recirculation and shear studies, the PCV15 formulationswere recirculated in a tubing loop. For the rotational studies, thePCV15base formulation (L-histidine, pH5.8and sodiumchloride)waspreparedand surfactants or stabilizerswereaddedasoutlined inExample7andTable 2. The agitation studies were designed using rotational side agitation for up 24 hours at 4°C. Visual assessment was used to evaluate the formulations. A path of a beam of light passing through the vessel allowed for the detection of particulates. Furthermore, the impact of manufacturing and shipping and handling stresses on particle size distribution was evaluated using static light scattering (SLS). Static light scattering of a suspension based drug product allows for themore sensitive detection of aggregation as indicated by particle size distribution. A monodisperse (monomodal) particle size distribution of a PCV15 drug product is indicative of a non-aggregated drug product. However a polydisperse, polymodal particle size distribution is indicative of aggregation. Stability of PCV15 in the stirring study

[0166] PCV15 formulations utilizing pneumococcal polysaccharide drug substances conjugated using reductive amination under aqueous conditions to CRM197 (referrred to as PCV15Aq), as described in Example 4, were screened using laboratory scale mixing experimental setup, as above (beaker and magnetic stir bar). PCV15Aq contains poly- saccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 using reductive amination under aqueous conditions. PCV15 in 20 mM L-histidine, 150 mM NaCl with APA was prepared in a 100 mL laboratory scale batch preparation using a beaker and magenetic stir bar. In the absence of a surfactant, thePCV15Aq formulationswereprone tomanufacture (stirring) induceddamage resulting in aggregation of the vaccine drug product during stirring to ensure homogeneity of the vaccine drug product.

[0167] Aclass of non-ionic triblock copolymerswere found to provide robust stability during screening studies of various excipients. Poloxamer 188 (P188) was selected to control aggregation and provide a robust and stable vaccine drug product formulation. PCV15Aq formulations were preparedwith 20mML-histidine, pH 5.8, 150mMNaCl, APA, and either no P188, 0.08% w / v P188, or 0.24% w / v P188. The impact of time under constant stirring using a magnetic stir bar was evaluated using static light scattering (Figures 6‑7). Particle size distribution was assessed using a Malvern Mastesizer 2000. A 5 µmNIST particle size standard was run and produced the expected size distribution. For all formulations (with and without Poloxamer 188), amonodisperse histogram profile was observed following addition of the conjugates to APA (T = 0 hr Stirring). In as little as 7 hours (T = 7 hr Stirring) of continuousmixing, the formulation without P188 resulted in the appearance of increased particle size distribution and aggregation (Figure 6).However, formulations containing P188 at either concentration showed no appearance of increased particle size or aggregation upon continuous mixing up to 24 hours (T = 24 hr Stirring) (Figure 7). 21 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 Stability of PCV15 in the horizontal agitation study

[0168] Additional PCV15Aq formulations in 20mML-histidine, pH 5.8, 150mMNaCl, and APA andwith or without 0.2% w / v P188 were prepared in a 100mL laboratory scale batch preparation as described in Example 7. To simulate shipping and handling and evaluate the impact to stability of the vaccine drug product, a horizontal agitation studywas utilized. The study represents a direct agitation of the PCV115Aq formulation through interactions with the surfaces in a container closuresystem(syringeor vial) andexposureof the formulation tofinal container componentsandanair interface. 0.64mL was dispensed into syringes and stoppered. These syringeswere horizontally rotated at 2‑8°C for 24 hours and evaluated for particle size distribution using SLS (Figure 8A). A visual assessment was also performed (Figure 8B). The PCV15Aq formulation in the absence of P188 and subjected to simulated shipping and handling stresses results in an increase in particle size distribution of the drug product and visible signs of agglomeration and aggregation with a container closure system such as a syringe. ThePCV15 formulationwith P188 did not show an increase in particle size distribution or visual signs of agglomeration and aggregation. EXAMPLE 9: Impact of Excipients on Stabilizing a Pneumococcal Conjugate Vaccine Drug Product Prepared Using a Mixture of Conjugates Generated by Reductive Amination in Aqueous and DMSO Solutions

[0169] Multiple 15-valent (PCV15) formulations with APA in 20 mM L-histidine, pH 5.8, 150 mM NaCl were evaluated using laboratory scale simulated shipping studies to ensure a robust manufacturable and commercially viable vaccine drugproduct formulation.FormulationPCV15Aqcontainedpneumococcal polysaccharide-CRM197conjugatesgenerated by reductive amination in aqueous solution. Formulation PCV15Aq / Non-Aq contained serotype 6A, 6B, 7F, 19A, 19F, and 23Fconjugatespreparedusing reductiveamination inDMSOandserotype1, 3, 4, 5, 9V,14, 18C,22F, and33Fconjugates prepared using reductive amination in aqueous solution, where all polysaccharides were conjugated to CRM197. Formulation PCV15Aq / Non-Aq / ST3-DTFB was similar to PCV15Aq / Non-Aq except that serotype 3 was conjugated to DTFB protein, not CRM197. Since the results were very similar between the two formulations at different concentrations of conjugates andAPA listed inExample7, only results forPCV15 formulation containing lower concentrations of conjugates and APA were shown in this Example, unless otherwise specified. Prevnar 13● was used as another example of a multivalent formulation to test the suitability of PS‑80 in a different pneumococcal conjugate vaccine product. It contains Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F polysaccharides conjugated to CRM197 carrier protein, polysorbate 80, succinatebuffer andaluminumphosphateadjuvant. Theconjugates areprepared using reductive amination using either DMSO or under aqueous conditions.

[0170] To evaluate the impact to stability of the vaccine formulations, a horizontal agitation studywas utilized. The study representsadirect agitationof formulations through interactionswith thesurfaces inacontainer closuresystem(syringeor vial) and exposure of the formulation to final container components and an air interface. Formulations were dispensed as 0.64mLfill into syringesand stoppered. These syringeswerehorizontally rotated at 2‑8°C for up to 8hr. The impact of time under horizontal agitation was evaluated for particle size distribution using static light scattering (SLS). Particle size and distribution were assessed using a Malvern Mastesizer 2000. A 5 µmNIST particle size standard was run and produced expected size distribution. As shown in Figure 9, P188 was not an effective stabilizer for controlling aggregation for the PCV15Aq / Non-Aq / ST3-DTFB formulation despite being a robust stabilizer for thePCV15Aq formulation. An increase in particle size distribution and visible signs of agglomeration and aggregation were noted in the PCV15Aq / Non-Aq / ST3-DTFB formula- tion with P188.

[0171] Due to the surprising discovery that P188 did not provide robust stability to the PCV15 formulation containing conjugates generated by reductive amination in DMSO, additional stabilizers and excipients were screened. PCV15Aq / - Non-Aq / ST3-DTFB formulations (withAPA in20mML-histidine, pH5.8, 150mMNaCl andvarious stabilizers)weredispensed into syringes and screenedusing horizontal agitation. The impact of timeunder constant horizontal rotationwasevaluated using visual assessment (Table 2). Table 2: Visual observation of PCV15Aq / Non-Aq / ST3-DTFB formulations containing 64 µg PnPs / mL and 250 µg / mL APA without surfactant, with P188, with PS‑80, or with PS‑20 after 1 hour of stirring and up to 24 hours of horizontal rotation in syringes. Fanning refers to the deposition of drug product formulation on the surface of the container closure system (e.g. syringe or vial) and is indicative of surface precipitation of the formulation. PCV15 Formulation No Rotation 1 hr Rotation 3 hr Rotation 6 hr Rotation 8.5 hr Rotation 24 hr Rotation No surfactant No aggrega- tion Slight Fan- ning Fanning Fanning Fanning Particulates 22 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) PCV15 Formulation No Rotation 1 hr Rotation 3 hr Rotation 6 hr Rotation 8.5 hr Rotation 24 hr Rotation 0.05% w / v P188 No aggrega- tion Fanning Fanning Fanning Not tested due to fanning at earlier time points0.1% w / v P188 No aggrega- tion Small fan- ning Small fanning Small fanning 0.5% w / v P188 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion 0.7% w / v P188 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion 1.0% w / v P188 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion 0.005% w / v PS‑80 No aggrega- tion Fanning Fanning Fanning Not tested due to fanning or particulates at earlier time points 0.01% w / v PS‑80 No aggrega- tion Fanning Fanning Fanning 0.05% w / v PS‑80 No aggrega- tion Fanning Fanning Fanning 0.005% w / v PS‑20 No aggrega- tion Fanning Fanning and small particu- lates Fanning and large particu- lates 0.01% w / v PS‑20 No aggrega- tion Fanning Fanning Fanning and large particu- lates 0.05% w / v PS‑20 No aggrega- tion No aggrega- tion No aggrega- tion Small fanning Small fan- ning Particulates 0.07% w / v No No No No aggrega- tion No No PS‑20 aggregation aggregation aggregation aggregation aggregation 0.1% w / v PS‑20 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion

[0172] Visual assessment ofPCV15Aq / Non-Aq / ST3-DTFB formulations indicatedno signof aggregationat higherP188and PS‑20 concentrations (Table 2). However, higher resolution studies to assess subvisible particle size distribution of PCV15Aq / Non-Aq / ST3-DTFB formulations containing P188 (0.05% w / v to 1.0% w / v) or PS‑20 (0.005% w / v - 0.1% w / v) were conducted using SLS. These formulations were horizontally rotated for up to 24 hours in 1.5mLHyPak syringes (Becton- Dickinson). D[4,3] values as measured by SLS are shown in Figure 10A-B. D[4,3] results show higher concentrations of PS‑20 significantly improved the physico-chemical stability of the formulation while P188 at all concentrations was not effective. As shown in Figure 10B, horizontal agitation of Prevnar 13® for up to 24 hrs showed an increase in D[4,3] values and the evidence of particulates indicating that the formulation was not sufficiently protected against simulated shipment induced aggregation and similar to our own experiences using PS‑80 in our formulations

[0173] Based on our data with PS‑20 and PS‑80, an improvement in stability would be expected with other pneumo- coccal polysaccharide-protein conjugates containing one or more conjugates produced in an aprotic solvent. EXAMPLE 10: Infant Rhesus Monkey (IRM) Immunogenicity Study using ST3-DTFB Conjugate Formulated in PCV15

[0174] DTFB prepared as described in Example 1 (multimodal cation exchange chromatography) was used to prepare ST3-DTFB conjugate as described in Example 3. PCV15 formulations were prepared as in Example 7. IRMs (Infant RhesusMonkeys, n=8 / group) were intramuscularly (Arms 1‑4) immunizedwith 100µL vaccine, as described in theTable 3 below on days 0, 28 and 56. Sera were collected prior to study start (pre) and on days 14, 28, 42, 56 and 70. IRMs were 23 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 observed twice daily by trained animal care staff for any signs of illness or distress. The vaccine formulations in IRMswere deemed to be safe and well tolerated, as no vaccine-related adverse events were noted. Table 3: Infant Rhesus Monkey study formulations Arm Formulation PnPs dose (µg) Conjugation process Protein carrier for serotype 3 1 PCV15Aq in 20 mM L-histidine, 150 mM NaCl, 0.2% w / v P188, 250 µg / mL APA 0.8 µg for 6B, 0.4 µg for all other sero- types All PnPs serotypes conju- gated to protein carrier in aqueous solution CRM197 2 PCV15Aq / Non-Aq in 20 mM L-histi- dine, 150 mM NaCl, 0.2% w / v P188, 250 µg / mL APA Serotypes 6A, 6B, 7F, 19A, 19F, 23F conjugated to protein carrier in DMSO; other serotypes conjugated to protein car- rier in aqueous solution 3 PCV15Aq / Non-Aq / ST3-DTFB in 20 mM L-histidine, 150 mM NaCl, 0.2% w / v P188, 250 µg / mL APA DTFB 4 PCV15Aq / Non-Aq / ST3-DTFB in20 mM L-histidine, 150 mM NaCl, 0.1% w / v PS‑20, 250 µg / mL APA

[0175] Mouse studies described in Example 6 were completed using monovalent conjugates with a single PnPs serotype 3WHOELISA completed to evaluate IgG responses. To assess serotype-specific IgG responses in a 15-valent vaccine, amultiplexed electrochemiluminescence (ECL) assay was developed for use with rhesusmonkey serum based on the human assay described byMarchese et al. usingMSD technology (MSD is a trademark ofMesoScale Discovery, a division of MesoScale Diagnostics, LLC., Gaithersburg, MD, U.S.A.) which utilizes a SULFO-TAG™ label that emits light upon electrochemical stimulation. Human antibody reagents and standards were used when testing the infant monkey samples. The infant rhesus monkey results were expressed as geometric mean concentrations read from a standard curve using the serotype-specific IgG concentrations assigned to the human reference standard (007sp).

[0176] Serotype 3 post-dose 3 (PD3) IgG responses (Figure 11) did not show any statistical differences among immunized groups. Functional antibody, evaluated in OPA assays using S. pneumoniae serotype 3 (Figure 12), did not show any show any statistical differences among immunized groups. EXAMPLE 11: Optimization of PCV15 Formulation

[0177] Since the results were very similar between the two formulations at different concentrations of conjugates and APA listed in Example 7, only results for PCV15 formulation containing lower concentrations of conjugates and APAwere shown in this Example, unless otherwise specified.

[0178] A recirculation line is used in the PCV15 formulation process to supply vaccine drug product to the syringe and vial fillingmachine. Recirculation during routine filling imparts additional shear and stress on biotherapeutic drug products and is therefore an important processing step to evaluate. Studies were conducted to evaluate the impact of recirculation on the PCV15Aq / Non-Aq / ST3-DTFB and formulations (in 20 mM L-histidine, pH 5.8, 150 mM NaCl, with 64 µg / mL total Polysaccharide and 250µg / mL APA) containing 0.2%w / v P188 and 0.1%w / v PS‑20. The formulations were recirculated for 24 hours froma feed container through tubing at a flow rate of 180mL / min using a peristaltic pump. The feed container was continuously mixed using a magnetic stir bar, and samples were periodically taken for visual observation. Formula- tions containing P188 showed the appearance of clumping or visual aggregation (Table 4),while formulations containing PS‑20 showed no signs of visible aggregation. Table 4: Visual assessment of PCV15 formulations containing 0.2% w / v P188 or 0.1% w / v PS‑20 during 24 hours of continuous mixing and recirculation PCV15 Formulation Recirculation time 0 hours 0.5 hours 1 hour 3 hours 6 hours 8 hours 12 hours 24 hours PCV15Aq / Non-Aq / ST3-DTFB with 0.2% w / v P188 No No No No No No No Yes 24 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 (continued) PCV15 Formulation Recirculation time 0 hours 0.5 hours 1 hour 3 hours 6 hours 8 hours 12 hours 24 hours PCV15Aq / Non-Aq / ST3-DTFB with 0.1% w / v PS‑20 No No No No No No No No No = No aggregation; Yes = Aggregation observed in recirculation bottle

[0179] Additional recirculation studies were conducted using PCV15Aq / Non-Aq and PCV15Aq / Non-Aq / ST3-DTFB formula- tions with up to 500 µg / mL (w / v Al+3) APA and with P188 or PS‑20. After 24 hours of recirculation with constant mixing, formulations were dispensed into syringes and horizontally agitated for up to 24 hours. The formulations were inpected, and a summary of the visual assessement for the syringes are shown in Table 5. These results indicate that PS‑20 provides a robust solution to physical instability or aggregation that may occur during routinemanufacturing and shipping and handling. P188 was unable to provide an adequate stability profile for this PCV15Aq / Non-Aq / ST3-DTFB formulation despite its success in stabilizing PCV15Aq formulations (Figures 7‑9). Table 5: Visual assessment of PCV15 formulations with 0.1% w / v PS‑20 or 0.2% w / v P188 after 24 hours of mixing and recirculation and up to 24 hours of horizontal rotation in 1.5 mL HyPak syringes PCV15 Formulation Horizontal rotation time after 24 hours of recirculation 0 hours 1 hour 3 hours 6 hours 10 hours 24 Hours PCV15Aq / Non-Aq / ST3-DTFB with 0.2% w / v P188 Small aggre- gates Small aggre- gates Small aggre- gates Large ag- gregates Extra large aggregates Extra large aggregates completely precipitated PCV15Aq / Non-Aq / ST3-DTFB with 0.1% w / v PS‑20 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion PCV15Aq / Non-Aq with 0.1% w / v PS‑20 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion PCV15Aq / Non-Aq with 0.2% w / v PS‑20 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion

[0180] An additional study was conducted on a PCV15 Aq / Non-Aq / ST3-DTFB formulation whereby ST18C was prepared using reductive amination in DMSO (PCV15 Aq / Non-Aq / ST3-DTFB / ST18C-Non-Aq). The PCV15 Aq / Non-Aq / ST3-DTFB / ST18C-Non-Aq formulationwasprepared in20mML-histidine, pH5.8, 150mMNaCl,with64µg / mL totalPolysaccharide, 250µg / mLAPA and 0.2% w / v PS‑20. After the PCV15 Aq / Non-Aq / ST3-DTFB / ST18C-Non-Aq formulation was recirculated for up to 6 hr with constantmixing, the formulationwas dispensed into syringes and horizontally agitated for up to 24 hours. The formulation was inspected, andasummaryof thevisual assessement for thesyringesare shown inTable6.These results indicate that PS‑20 provides a robust solution to physical instability or aggregation that may occur during routine manufacturing and shipping and handling with a formulation comprising a PCV15 formulation prepared in 20mML-histidine, pH 5.8, 150mM NaCl, with 250µg / mLAPAand0.2%w / vPS‑20 and containing pneumococcal polysaccharide serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugates prepared using reductive amination in DMSO and serotype 1, 3, 4, 5, 9V, 14, 22F, and 33F conjugates prepared using reductive amination in aqueous solution, where all polysaccharides were conjugated to CRM197. Table 6: Visual assessment of PCV15 formulations with 0.2% w / v PS‑20 after 24 hours of mixing and recirculation and up to 24 hours of horizontal rotation in 1.5 mL HyPak syringes PCV15 Formulation Horizontal rotation time after 24 hours of recirculation 0 hours 8 hour 16 hours 24 Hours PCV15Aq / Non-Aq / ST3-DTFB / ST18C Non-Aq with 0.2% PS20 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion 25 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55

[0181] As shown in Figure 10 and Tables 4‑5, P188 alone provided minimal benefit in preventing aggregation of suspension-based PCV15 formulations composed of conjugates generated using reductive amination in DMSO and / or ST3-DTFBconjugate.Additionalmixingandhorizontal rotation studieswere conductedwith formulations containing0.2% w / v P188 and PEG400 (PEG) or Propylene Glycol (PG) stabilizers. PCV15Aq / Non-Aq / ST3-DTFB formulations were prepared at 500 mL scale as described in Example 7. PEG400 or PG was added to the APA, and conjugates were then added. The final concentration of PEG400 or PG was between 0% w / v to 15% w / v in the formulation containing 64 µg PnPs / mL, 250 µg / mL APA, and 0.2% w / v P188. Formuations were mixed continuously for 1 hour with a magnetic stir bar and were dispensed into syringes. Syringes were hortizontally agitated for up to 24 hours. The formulations were periodically sampled for visual assessment and for particle size distributionmeasurements by SLS. Results from visual assessement of syringes are shown inTables 7‑8.Particle size distribution results are shown inFigure 13.Whenaddedalone, P188did not control aggregation in the formulation. Surprisingly, PEG400 or PG provided adequate stability when combined with P188. Table 7: Visual assessment of PCV15 formulations with 0.2% w / v P188 and various PEG400 concentrations after 1 hour of mixing and up to 24 hours of horizontal rotation in 1.5 mL HyPak syringes PCV15Aq / Non-Aq / ST3-DTFB with 0.2% w / v P188 Horizontal rotation time after 1 hour of mixing 0 Hours 3 hour 6 hours 18 hours 24 Hours with 0% PEG400 No aggrega- tion No aggrega- tion Small aggre- gates Large aggre- gates; fan- ning Large aggregates; considerable fan- ning with 6% PEG400 No aggrega- tion No aggrega- tion No aggrega- tion Slight fanning ring Small aggregates; Slight fanning with 8% PEG400 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion Slight fanning ring with 10% PEG400 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggregation with 12% PEG400 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggregation with 15% PEG400 No aggrega- tion No aggrega- tion No aggrega- tion No aggrega- tion No aggregation Table 8: Visual assessment of PCV15 formulations with 0.2% w / v P188 and various PG concentrations after 1 hour of mixing and up to 24 hours of horizontal rotation in 1.5 mL HyPak syringes PCV15Aq / Non-Aq / ST3-DTFB with 0.2% w / v P188 Horizontal rotation time after 1 hour of mixing 0 Hours 3 hour 6 hours 18 hours 24 Hours with 0% PG No aggrega- tion No aggrega- tion Small aggre- gates Large aggre- gates; slight fanning Large aggre- gates; fanning with 8% PG No aggrega- tion No aggrega- tion No aggrega- tion No aggregation Small aggre- gates with 10% PG No aggrega- tion No aggrega- tion No aggrega- tion No aggregation Noaggregation with 15% PG No aggrega- tion No aggrega- tion No aggrega- tion No aggregation Noaggregation Example 12: Immunogenicity of Stabilizing Formulations of Pneumococcal Conjugate Vaccine

[0182] The impact of PCV15 containing formulations that had been optimized to control manufacturing and shipping induced instability on Infant Rhesus Monkey immunogenicity was assessed. Eight animals per group received an intramuscular injectionwith 0.1mLofPCV15Aq / Non-Aq / ST3-DTFB formulation containing 64µgPnPs / mL, 20mML-histidine, pH5.8, 150mMsodiumchloride, 250µg / mLAPA, andeither 0.2%w / vP188, 15%w / vPGor 0.1%w / vPS‑20as described 26 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 in Example 11. Injectionswere administered at T=0 (Dose 1), 1month (Dose 2) and 2months (Dose 3) of age. Serumwas collected prior to Dose 1 and 2weeks post dose 1, 2, and 3. The serum IgG levels from the pre-immune, post dose‑1, post dose‑2, andpost dose‑3serumsamplesweredeterminedasdescribedabove inExample10. The results shown inFigure 14 indicate that PCV15 formulations with either PS‑20 or a combination of P188 and PG were immunogenic. EXAMPLE 13: Impact of Polysorbate 20 and Polysorbate 80 on Stabilizing a Pneumococcal Conjugate Vaccine Drug Product Prepared Using Different Mixtures of Conjugates Generated by Reductive Amination in protic (Aqueous) and aprotic (DMSO) solutions

[0183] The promising results seen with the PCV15 formulations in the above Examples warranted exploration of the lower and upper limits of the ratio of protein levels in glycoconjugates made in DMSO vs. aqueous conditions. Multiple polyvalent PCV formulations havingdifferent ratios of glycoconjugates containedpneumococcal polysaccharide-CRM197 conjugates generated by reductive amination in an aqueous solution or inDMSO.Each formulationwas normalized to the amount of protein and contained a total Polysaccharide (Ps) concentration of 64µg / mL (w / vPs)with 250µg / mLAPA in 20 mM L-histidine, pH 5.8, 150 mM NaCl. Either PS‑80 or PS‑20 was added to each formulation to achieve a final concentration of either PS‑80 (0.05% w / v) or PS‑20 (0.05% w / v) or PS‑20 (0.2% w / v). The formulations were dispensed into BD HyPAK Prefilled syringes and evaluated using laboratory scale simulated shipping studies to ensure a robust manufacturable and commercially viable vaccine drug product formulation.

[0184] To achieve the desired range of percentages of glycoconjugates prepared using DMSO, the following formula- tions were prepared where all polysaccharides were conjugated to CRM197 using reductive amination: Formulation PCV24% contained serotypes 6A, 6B and 23F conjugates prepared inDMSO, and serotypes 1, 3, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, and 33F conjugates prepared in an aqueous solution. The total protein in this formulation was 56 µg / mL with 13 µg / mL or ~24% of the total protein consisting of CRM197 conjugated to polysaccharide using reductive amination in DMSO.

[0185] Formulation PCV50% contained serotypes 6A, 6B, 7F, 19A, 19F and 23F conjugates prepared in DMSO, and serotypes 1, 3, 4, 5, 9V, 14, 18C, 22F, and 33F conjugates prepared in an aqueous solution. The total protein in this formulation was 62 µg / mL with 31 µg / mL or 50% of the total protein consisting of CRM197 conjugated to polysaccharide using reductive amination in DMSO.

[0186] Formulation PCV62% contained serotypes 6A, 6B, 7F, 19A, 19F and 23F conjugates prepared in DMSO, and serotypes 1, 5, 9V, 14, 18C, 22F, and 33F conjugates prepared in an aqueous solution. The total protein in this formulation was 61 µg / mL with 38 µg / mL or ~62% of the total protein consisting of CRM197 conjugated to polysaccharide using reductive amination in DMSO.

[0187] Formulation PCV79% contained serotypes 6A, 6B, 7F, 19A, 19F and 23F conjugates prepared in DMSO, and serotypes 1, 5, 18C, and 33F conjugates prepared in an aqueous solution. The total protein in this formulation was 63 µg / mL with 50 µg / mL or ~79% of the total protein consisting of CRM197 conjugated to polysaccharide using reductive amination in DMSO.

[0188] Formulation PCV100% contained serotypes 6A, 6B, 7F, 19A, 19F and 23F conjugates prepared using reductive amination inDMSO.The total protein in this formulationwas65µg / mLwith65µg / mLor100%of the total protein consisting of CRM197 conjugated to polysaccharide in DMSO.

[0189] A horizontal agitation study was utilized to evaluate the impact on product stability of the ratio of protein conjugated to polysaccharide in DMSO to the total protein. The study represents a direct agitation of formulations through interactions with the surfaces in a container closure system (syringe or vial) and exposure of the formulation to final container components and an air interface. Formulations were dispensed as 0.64 mL fill into syringes and stoppered. These syringes were horizontally rotated at 2‑8°C for up to 24 hr. The impact of time under horizontal agitation was evaluated for particle sizedistributionusing static light scattering (SLS). Particle sizeanddistributionwereassessedusing a Malvern Mastesizer 2000. A 5 µm NIST particle size standard was run and produced expected size distribution. As shown inFigures15A-E, PS‑80wasnotaneffectivestabilizer for controllingaggregation forall of thePCVcontainingDrug products (PCV24% toPCV100%). An increase in particle size distribution and visible signs of agglomeration (appearance of particles) and aggregation were observed for all the formulations with PS‑80.

[0190] Surprisingly, PS‑20 at a comparable concentration to that used for PS‑80 provided an improved stability profile across the range of DMSO conjugate percentages tested. Adding PS‑20 to achieve a concentration of 0.2% PS‑20 in the formulation buffer resulted in superior stability compared to 0.05% PS‑20 across the range of DMSO conjugate percentages tested. Example 14: Immunogenicity of Stabilizing Formulations of Pneumococcal Conjugate Vaccine in New Zealand White Rabbits

[0191] The impact of PCV15 containing formulations that had been optimized to control manufacturing and shipping 27 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 induced instability on New Zealand White Rabbit immunogenicity was assessed. Eight animals per group received an intramuscular injection with 0.1 mL of PCV15Aq / Non-Aq formulation containing 64 mg PnPs / mL, 20 mM L-histidine, pH 5.8, 150 mM sodium chloride, 250 µg / mL APA, and 0.2% w / v PS‑20 as described in Example 11. Injections were administeredatday0and14.Serumwascollectedprior to vaccinationondays0and14andalsoonday28.TheserumIgG levels from the pre-immune, post dose‑1, post dose‑2 serum samples were determined by ECL analysis.

[0192] The results shown inFigure 16 indicate that a 15-valent pneumococcal conjugate formulation in 20mMHistidine pH 5.8, 150 mM NaCl, 250 µg / mL APA, 0.2% w / v PS‑20 having S. pneumoniae polysaccharides from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugated to CRM197 using reductive amination in DMSO and S. pneumoniae polysacchar- ides from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F conjugated to CRM197 using reductive amination in aqueous solution formulated as a dosage form containing 4 µg / mL of each saccharide, except for 6B at 8 µg / mL; and about 64 µg / mL CRM197 carrier protein, is immunogenic. WHAT IS FEATURED IS:

[0193] 1.A formulationcomprising (i) oneormorepolysaccharide-protein conjugates; (ii) apHbufferedsalinesolutionhaving apH in the range from5.0 to7.5; (ii) analuminumsalt; and (iv) a surfactant systemselected froma) polysorbate20and (b) a poloxamer having a molecular weight in the range from 1100 Da to 17,400 Da and a polyol selected from propylene glycol and polyethylene glycol 400. 2. The formulation of feature 1, wherein one or more of the polysaccharide-protein conjugates are made in an aprotic solvent. 3. The formulation of feature 1, wherein 10 to 100% of the polysaccharide-protein conjugates by weight protein are made in an aprotic solvent. 4. The formulation of feature 2 or 3, wherein the aprotic solvent is DMSO. 5. The formulationof feature 1,wherein the surfactant systemcomprisesapoloxamerwhich hasamolecularweight in the range from 1100 Da to 17,400 Da. 6. The formulation of feature 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 15,000 Da. 7. The formulation of feature 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 10,000 Da. 8. The formulation of any one of features 1 to 4, wherein the poloxamer is poloxamer 188 or poloxamer 407. 9. The formulation of any one of features 1 to 8, wherein the final concentration of the poloxamer is from0.001% to 5% weight / volume. 10.The formulationof feature9,wherein thefinal concentrationof thepoloxamer is from0.025%to1%weight / volume. 11. The formulation of any one of features 1 to 10, wherein the polyol is propylene glycol and is at final concentration from 1% to 20% weight / volume. 12. The formulation of any one of features 1 to 10, wherein the polyol is polyethylene glycol 400 and is at final concentration from 1% to 20% weight / volume. 13. The formulation of feature 1, wherein the surfactant system comprises polysorbate 20. 14. The formulation of feature 13, wherein the final concentration of the polysorbate 20 is in the range from 0.001% to 10% weight / volume. 15. The formulation of feature 13, wherein the final concentration of the polysorbate 20 is in the range from 0.025% to 2.5% weight / volume. 28 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 16. The formulation of feature 13, wherein the final concentration of the polysorbate 20 is in the range from 0.025% to 0.1% weight / volume. 17. The formulation of any one of features 13 to 16, further comprising a polyol selected from propylene glycol and polyethylene glycol. 18. The formulation of feature 17, wherein the polyethylene glycol or propylene glycol is at a final concentration of 6% to 20% weight / volume. 19. The formulation of feature 18, wherein the polyethylene glycol is polyethylene glycol 400. 20. The formulation of any one of features 1 to 19, wherein the pH buffered saline solution has a pH in the range from 5.0 to 7.0. 21. The formulation of feature 20, wherein the buffer selected from the group consisting of phosphate, succinate, L- histidine, MES, MOPS, HEPES, acetate and citrate. 22. The formulation of feature 21, wherein the buffer is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM. 23. The formulation of feature 22, wherein the L-histidine is at a final concentration of 20 mM ± 2 mM. 24. The formulation of any one of features 1 to 23, wherein the salt in the pH buffered saline solution is magnesium chloride, potassium chloride, sodium chloride or a combination thereof. 25. The formulation of feature 24, wherein the salt in the pH buffered saline solution is sodium chloride. 26. The formulation of any one of features 1 to 25, wherein the saline is present at a concentration from 20mM to 170 mM. 27. The formulation of any one of features 1 to 26, wherein the polysaccharide-protein conjugates comprise one or more pneumococcal polysaccharides conjugated to a carrier protein. 28. The formulation of feature 27, wherein the carrier protein is selected from CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof. 29. The formulation of feature 28, wherein one or more of the polysaccharide-protein conjugates are conjugated to CRM197. 30. The formulation of feature 29, wherein the polysaccharide-protein conjugate formulation is a 15-valent pneu- mococcal conjugate (15vPnC) formulation consisting essentially ofS. pneumoniae polysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated to CRM197. 31. The formulation of feature 30, wherein one or more of the polysaccharide protein conjugates are prepared using reductive amination under DMSO conditions. 32. The formulation of feature 31, wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous conditions. 33. The formulationof feature 32,wherein eachdose is formulated to contain: 4µg / mLor 8µg / mLof each saccharide, except for 6B at 8 µg / mL or 16 µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein. 34. The formulation of feature 33, further comprising 20mML-histidine, pH5.8, 150mMsodiumchloride, 0.25mg / mL of Aluminum Phosphate Adjuvant (APA) and 0.2% w / v PS‑20. 35. A formulation comprising a 15-valent pneumococcal conjugate composition consisting essentially of S. pneu- 29 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 moniae polysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated to CRM197; 20mMHistidine pH 5.8; 150mMNaCl; 250 µg / mL APA; and 0.2%w / v PS‑20; formulated as a dosage form containing 4 µg / mL of each saccharide, except for 6B at 8 µg / mL; and about 64 µg / mL CRM197 carrier protein; whereinpolysaccharideprotein conjugates fromserotypes6A,6B,7F,18C,19A,19F,and23FpreparedunderDMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F prepared using aqueous conditions. Claims 1. A formulation comprising (i) oneormorepolysaccharide-protein conjugates; (ii) a pHbuffered saline solution having a pH in the range from 5.0 to 7.5; (ii) an aluminum salt; and (iv) a surfactant system selected from a) polysorbate 20 and (b) a poloxamer having a molecular weight in the range from 1100 Da to 17,400 Da and a polyol selected from propylene glycol and polyethylene glycol 400. 2. The formulation of claim 1, wherein one or more of the polysaccharide-protein conjugates are made in an aprotic solvent. 3. The formulationof claim1,wherein10 to100%of thepolysaccharide-proteinconjugatesbyweightproteinaremade in an aprotic solvent. 4. The formulation of claim 2 or 3, wherein the aprotic solvent is DMSO. 5. The formulation of claim 1,wherein the surfactant systemcomprises a poloxamerwhich has amolecular weight in the range from 1100 Da to 17,400 Da. 6. The formulation of claim 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 15,000 Da. 7. The formulation of claim 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 10,000 Da. 8. The formulation of any one of claims 1 to 4, wherein the poloxamer is poloxamer 188 or poloxamer 407. 9. The formulation of any one of claims 1 to 8, wherein the final concentration of the poloxamer is from 0.001% to 5% weight / volume. 10. The formulation of claim 9, wherein the final concentration of the poloxamer is from 0.025% to 1% weight / volume. 11. The formulationof anyoneof claims1 to10,wherein thepolyol ispropyleneglycol and isat final concentration from1% to 20% weight / volume. 12. The formulation of any one of claims 1 to 10, wherein the polyol is polyethylene glycol 400 and is at final concentration from 1% to 20% weight / volume. 13. The formulation of claim 1, wherein the surfactant system comprises polysorbate 20. 14. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from 0.001% to 10% weight / volume. 15. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from0.025% to 2.5% weight / volume. 16. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from0.025% to 0.1% weight / volume. 17. The formulation of any one of claims 13 to 16, further comprising a polyol selected from propylene glycol and polyethylene glycol. 18. The formulationof claim17,wherein thepolyethyleneglycol or propyleneglycol is at afinal concentrationof 6%to20% 30 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 weight / volume. 19. The formulation of claim 18, wherein the polyethylene glycol is polyethylene glycol 400. 20. The formulation of any one of claims 1 to 19, wherein the pH buffered saline solution has a pH in the range from 5.0 to 7.0. 21. The formulation of claim 20, wherein the buffer selected from the group consisting of phosphate, succinate, L- histidine, MES, MOPS, HEPES, acetate and citrate. 22. The formulationof claim21,wherein thebuffer is L-histidineat a final concentrationof 5mM to50mM,or succinateat a final concentration of 1 mM to 10 mM. 23. The formulation of claim 22, wherein the L-histidine is at a final concentration of 20 mM ± 2 mM. 24. The formulation of any one of claims 1 to 23, wherein the salt in the pHbuffered saline solution ismagnesium chloride, potassium chloride, sodium chloride or a combination thereof. 25. The formulation of claim 24, wherein the salt in the pH buffered saline solution is sodium chloride. 26. The formulation of any one of claims 1 to 25, wherein the saline is present at a concentration from 20mM to 170mM. 27. The formulation of any one of claims 1 to 26, wherein the polysaccharide-protein conjugates comprise one or more pneumococcal polysaccharides conjugated to a carrier protein. 28. The formulation of claim 27, wherein the carrier protein is selected fromCRM197, diphtheria toxin fragment B (DTFB), DTFBC8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid,E. coli LT,E. coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof. 29. The formulationof claim28,whereinoneormoreof thepolysaccharide-protein conjugatesareconjugated toCRM197. 30. The formulation of claim 29, wherein the polysaccharide-protein conjugate formulation is a 15-valent pneumococcal conjugate (15vPnC) formulation consisting essentially ofS.pneumoniaepolysaccharide fromserotypes1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated to CRM197. 31. The formulation of claim 30, wherein one or more of the polysaccharide protein conjugates are prepared using reductive amination under DMSO conditions. 32. The formulation of claim 31, wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous conditions. 33. The formulation of claim 32, wherein each dose is formulated to contain: 4 µg / mL or 8 µg / mL of each saccharide, except for 6B at 8 µg / mL or 16 µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein. 34. The formulation of claim 33, further comprising 20 mM L-histidine, pH 5.8, 150 mM sodium chloride, 0.25 mg / mL of Aluminum Phosphate Adjuvant (APA) and 0.2% w / v PS‑20. 35. A formulation comprising a 15-valent pneumococcal conjugate composition consisting essentially of S. pneumoniae polysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated toCRM197; 20mMHistidine pH5.8; 150mMNaCl; 250µg / mLAPA; and0.2%w / vPS‑20; formulated as adosage formcontaining 4 µg / mL of each saccharide, except for 6B at 8 µg / mL; and about 64 µg / mL CRM197 carrier protein; wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F prepared using aqueous conditions. 31 EP 4 656 650 A2 5 10 15 20 25 30 35 40 45 50 55 32 EP 4 656 650 A2 33 EP 4 656 650 A2 34 EP 4 656 650 A2 35 EP 4 656 650 A2 36 EP 4 656 650 A2 37 EP 4 656 650 A2 38 EP 4 656 650 A2 39 EP 4 656 650 A2 40 EP 4 656 650 A2 41 EP 4 656 650 A2 42 EP 4 656 650 A2 43 EP 4 656 650 A2 44 EP 4 656 650 A2 45 EP 4 656 650 A2 46 EP 4 656 650 A2 47 EP 4 656 650 A2 48 EP 4 656 650 A2 49 EP 4 656 650 A2 50 EP 4 656 650 A2 51 EP 4 656 650 A2 52 EP 4 656 650 A2 53 EP 4 656 650 A2 54 EP 4 656 650 A2 REFERENCES CITED IN THE DESCRIPTION This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard. Patent documents cited in the description • US 20060228380 A1

[0005] • CN 101590224 A

[0006] • US 8192746 B

[0007] • US 20100209450

[0008] • US 20100074922 A

[0008] • US 20090017059 A

[0008] • US 20090010959 A

[0008] • US 20090017072 A

[0008] • US 8562999 B

[0009] • US 20130273098

[0009] • US 5614382 A

[0034] • WO 2004083251 A

[0035] • WO 02091998 A

[0035] • WO 04081515 A

[0035] • WO 0198334 A

[0035] • WO 0354007 A

[0035] • EP 0594610 A

[0035] • EP 0378881 A

[0035] • EP 0427347 A

[0035] • WO 9317712 A

[0035] • WO 9403208 A

[0035] • WO 9858668 A

[0035] • EP 0471177 A

[0035] • WO 9101146 A

[0035] • WO 0172337 A

[0035] • WO 0061761 A

[0035] • US 4709017 A

[0036] • US 4950740 A

[0036] • US 5917017 A

[0036] • US 6455673 B

[0036] • US 5843711 A

[0036] • EP 497524 A

[0042] • EP 497525 A

[0042] • US 20080286838

[0042] • US 5847112 A

[0042]

[0126] • US 4365170 A

[0044] • US 4673574 A

[0044] • US 4902506 A

[0044] • US 20150231270 A1

[0045] • EP 0471177 B1

[0045] • US 20110195086 A1

[0045] • WO 9315760 A

[0050] • WO 9508348 A

[0050] • WO 9629094 A

[0050] • WO 9842721 A

[0051] • US 6146902 A

[0052] • WO 9014837 A

[0057] • US 4912094 A

[0057] • US 5057540 A

[0057] • US 6113918 A

[0057] • US 6207646 B

[0057] • WO 02083855 A

[0078]

[0109] • WO 02053761 A

[0078]

[0109] • US 3740421 A

[0100] • WO 2012173876 A1

[0112]

[0142]

[0153] • EP 0497524 A

[0126] • US 20110195086

[0130] • US 5623057 A, Marburg

[0133] Non-patent literature cited in the description • ARDITI et al. Pediatrics, 1998, vol. 102, 1087-97

[0002] • Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep, 2005, vol. 54 (36)

[0004] • O’BRIEN et al. Am J Epidemiol, 2004, vol. 159, 634- 44

[0004] • WHITNEYet al. N Engl J Med, 2003, vol. 348, 1737- 46

[0004] • KYAW et al. N Engl J Med, 2006, vol. 354, 1455-63

[0004] • HICKS et al. J Infect Dis, 2007, vol. 196, 1346-54

[0004] • TRAORE et al. Clin Infect Dis, 2009, vol. 48, S181- S189

[0004] • PRYMULA et al. Lancet, 2006, vol. 367, 740-48

[0005] • KIENINGER et al. Safety and Immunologic Non- inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Con- jugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers. 48th Annual ICAAC / ISDA 46th Annual Meeting, 2008

[0005] • DAGANetal. Infect Immun, 1998, vol. 66, 2093-2098

[0005] • FATTOM. Vaccine, 1999, vol. 17, 126

[0005] • GILL, D. M. ; DINIUS, L. L. J. Biol. Chem., 1971, vol. 246, 1485-1491

[0026] • GILL, D. M. ; PAPPENHEIMER, JR., A. M. J. Biol. Chem., 1971, vol. 246, 1492-1495

[0026] • COLLIER, R. J. ; KANDEL, J. J. Biol. Chem., 1971, vol. 246, 1496-1503

[0026] 55 EP 4 656 650 A2 • DRAZIN, R. ; KANDEL, J. ; COLLIER, R. J. J. Biol. Chem., 1971, vol. 246, 1504-1510

[0026] • GREENFIELD, L. ; BJORN, M.J. ; HORN, G. ; FONG,D. ;BUCK,G.A. ;COLLIER, R.J. ;KAPLAN, D.A. Proc. Natl. Acad. Sci. USA, 1983, vol. 80, 6853- 6857

[0027] • BIXLER et al. Adv Exp Med Biol., 1989, vol. 251

[0028] • RAJU et al. Eur. J. Immunol., 1995, vol. 25, 3207- 3214

[0028]

[0029] • DIETHELM-OKITA et al. J Infect Dis, 2000, vol. 181, 1001-9

[0028] • MCCOOLetal. Infect. and Immun., September1999, vol. 67, 4862-4869

[0028] • BIXLERetal.AdvExpMedBiol., 1989, vol. 251, 175- 80

[0029] • KUO et al. Infect Immun, 1995, vol. 63, 2706-13

[0035] • FALUGI et al. Eur J Immunol, 2001, vol. 31, 3816- 3824

[0035] • BARALDOIet al. Infect Immun, 2004, vol. 72, 4884-7

[0035] • BEN-YEDIDIA et al. Immunol Lett, 1998, vol. 64, 9

[0035] • UCHIDA et al. J Biol Chem, 1973, vol. 218, 3838- 3844

[0036] • GIDLEY et al. Biochem J., 1982, vol. 203, 331-334

[0047] • JENTOFTet al. Anal Biochem., 1980, vol. 106, 186- 190

[0047] • CHU et al. Infect. Immunity, 1983, vol. 40, 245-256

[0050] • BETHELL et al. J. Biol. Chem., 1979, vol. 254, 2572- 4

[0051] • HEARN et al. J. Chromatogr., 1981, vol. 218, 509-18

[0051] • HARLOW, E. ; D. LANE. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1988

[0060] • NICKLAS, W. Aluminum salts. Research in Immu- nology, 1992, vol. 143, 489-493

[0060] • LAMBRECHT et al. Curr Opin Immunol, 2009, vol. 21, 23

[0062] • WANG et al. Vaccine, 2003, vol. 21, 4297

[0065] • SURetal.J Immunol., 1999, vol. 162,6284-93

[0066] • VERTHELYI.MethodsMol Med, 2006, vol. 127, 139- 58

[0066] • YASUDA et al. Crit Rev Ther Drug Carrier Syst., 2006, vol. 23, 89-110

[0066] • ANDERSONet al. J. Immunol., 1986, vol. 137, 1181- 1186

[0130] • LANDER et al. Biotechnol. Prog., 2000, vol. 16, 80- 85

[0133] 摘要 本發明提供肺炎球菌結合疫苗製劑，所述製劑包含表面活性劑體 系，該體系含有聚山梨酯 20，或泊洛沙姆與多元醇的組合物。

Claims

1. A formulation comprising (i) one or more polysaccharide-protein conjugates; (ii) a pH buffered saline solution having a pH in the range from 5.0 to 7.5; (ii) an aluminum salt; and (iv) a surfactant system selected from a) polysorbate 20 and (b) a poloxamer having a molecular weight in the range from 1100 Da to 17,400 Da and a polyol selected from propylene glycol and polyethylene glycol 400.

2. The formulation of claim 1, wherein one or more of the polysaccharide-protein conjugates are made in an aprotic solvent.

3. The formulation of claim 1, wherein 10 to 100% of the polysaccharide-protein conjugates by weight protein are made in an aprotic solvent.

4. The formulation of claim 2 or 3, wherein the aprotic solvent is DMSO.

5. The formulation of claim 1, wherein the surfactant system comprises a poloxamer which has a molecular weight in the range from 1100 Da to 17,400 Da.

6. The formulation of claim 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 15,000 Da.

7. The formulation of claim 5, wherein the poloxamer has a molecular weight in the range from 7,500 Da to 10,000 Da.

8. The formulation of any one of claims 1 to 4, wherein the poloxamer is poloxamer 188 or poloxamer 407.

9. The formulation of any one of claims 1 to 8, wherein the final concentration of the poloxamer is from 0.001% to 5% weight / volume.

10. The formulation of claim 9, wherein the final concentration of the poloxamer is from 0.025% to 1% weight / volume.

11. The formulation of any one of claims 1 to 10, wherein the polyol is propylene glycol and is at final concentration from 1% to 20% weight / volume.

12. The formulation of any one of claims 1 to 10, wherein the polyol is polyethylene glycol 400 and is at final concentration from 1% to 20% weight / volume.

13. The formulation of claim 1, wherein the surfactant system comprises polysorbate 20.

14. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from 0.001% to 10% weight / volume.

15. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from 0.025% to 2.5% weight / volume.

16. The formulation of claim 13, wherein the final concentration of the polysorbate 20 is in the range from 0.025% to 0.1% weight / volume.

17. The formulation of any one of claims 13 to 16, further comprising a polyol selected from propylene glycol and polyethylene glycol.

18. The formulation of claim 17, wherein the polyethylene glycol or propylene glycol is at a final concentration of 6% to 20% weight / volume.

19. The formulation of claim 18, wherein the polyethylene glycol is polyethylene glycol 400.

20. The formulation of any one of claims 1 to 19, wherein the pH buffered saline solution has a pH in the range from 5.0 to 7.0.

21. The formulation of claim 20, wherein the buffer selected from the group consisting of phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate and citrate.

22. The formulation of claim 21, wherein the buffer is L-histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM.

23. The formulation of claim 22, wherein the L-histidine is at a final concentration of 20 mM ± 2 mM.

24. The formulation of any one of claims 1 to 23, wherein the salt in the pH buffered saline solution is magnesium chloride, potassium chloride, sodium chloride or a combination thereof.

25. The formulation of claim 24, wherein the salt in the pH buffered saline solution is sodium chloride.

26. The formulation of any one of claims 1 to 25, wherein the saline is present at a concentration from 20 mM to 170 mM.

27. The formulation of any one of claims 1 to 26, wherein the polysaccharide-protein conjugates comprise one or more pneumococcal polysaccharides conjugated to a carrier protein.

28. The formulation of claim 27, wherein the carrier protein is selected from CRM197, diphtheria toxin fragment B (DTFB), DTFB C8, Diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof.

29. The formulation of claim 28, wherein one or more of the polysaccharide-protein conjugates are conjugated to CRM197.

30. The formulation of claim 29, wherein the polysaccharide-protein conjugate formulation is a 15-valent pneumococcal conjugate (15vPnC) formulation consisting essentially of S. pneumoniae polysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated to CRM197.

31. The formulation of claim 30, wherein one or more of the polysaccharide protein conjugates are prepared using reductive amination under DMSO conditions.

32. The formulation of claim 31, wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous conditions.

33. The formulation of claim 32, wherein each dose is formulated to contain: 4 µg / mL or 8 µg / mL of each saccharide, except for 6B at 8 µg / mL or 16 µg / mL; and about 64 µg / mL or 128 µg / mL CRM197 carrier protein.

34. The formulation of claim 33, further comprising 20 mM L-histidine, pH 5.8, 150 mM sodium chloride, 0.25 mg / mL of Aluminum Phosphate Adjuvant (APA) and 0.2% w / v PS-20.

35. A formulation comprising a 15-valent pneumococcal conjugate composition consisting essentially of S. pneumoniae polysaccharide from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23 F and 33F conjugated to CRM197; 20 mM Histidine pH 5.8; 150 mM NaCl; 250 µg / mL APA; and 0.2% w / v PS-20; formulated as a dosage form containing 4 µg / mL of each saccharide, except for 6B at 8 µg / mL; and about 64 µg / mL CRM197 carrier protein; wherein polysaccharide protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F prepared under DMSO conditions and polysaccharide protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F prepared using aqueous conditions.

Images