Immunogenic serotype 35b pneumococcal polysaccharide-protein conjugate and conjugation process for making the same

Abstract

To provide an improved process related to the conjugation of capsular polysaccharides from Streptococcus pneumoniae (S. pneumoniae) serotype 35B to a carrier protein, and a prepared serotype 35B polysaccharide-protein conjugate.SOLUTION: A serotype 35B S. pneumoniae polysaccharide-protein conjugate has a molecular weight of 1,000 kDa to 7,000 kDa. The conjugate comprises a lysine consumption of 3 mol / mol protein to 9 mol / mol protein. A process for making the conjugate comprises activation of the polysaccharide, wherein the activation utilizes periodate in a range of 0.01 to 0.1 moles of periodate per mole of polysaccharide repeating unit.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention provides an improvement in a process related to the conjugation of capsular polysaccharides from Streptococcus pneumoniae (S. pneumoniae) serotype 35B to carrier proteins of the same class. The serotype 35B polysaccharide-carrier protein conjugate prepared by the disclosed process is, inter alia, more immunogenic than similar conjugates prepared by prior art methods. The Streptococcus pneumoniae serotype 35B polysaccharide-carrier protein conjugate prepared using the process of the present invention can be included in a multivalent pneumococcal conjugate vaccine composition.

Background Art

[0002] Streptococcus pneumoniae, an example of an encapsulated bacterium, is an important cause of serious diseases worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated that in the United States 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 per year. See "Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46(RR-8):1-13". Furthermore, the complications of these diseases can be serious, and some studies have reported a mortality rate of up to 8% and neurological sequelae of 25% due to pneumococcal meningitis. See "Arditi et al., 1998, Pediatrics 102:1087-97".

[0003] ​​​​​​​​​​​​​​The polyvalent pneumococcal polysaccharide vaccine that has been approved for many years has been found to be very valuable in the prevention of pneumococcal diseases in adults, especially the elderly and high-risk adults. However, infants and young children have an insufficient response to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell-independent immunogens, and in infants, the response is weak or not induced at all. Chemical conjugation of bacterial polysaccharide immunogens to carrier proteins converts the immune response to T-cell dependence in infants. Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM197 are described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T-cell stimulating epitopes in their amino acid sequences. At that time, a pneumococcal conjugate vaccine, Prevnar®, containing the seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) most frequently isolated from causing invasive pneumococcal diseases in infants and young children was first approved in the United States in February 2000. After Prevnar® was widely used in the United States, the invasive pneumococcal diseases in children were significantly reduced by the serotypes present in Prevnar®. See "Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2 005, 54(36):893-7". However, in certain regions of the world, there are limitations to the range of serotypes by Prevnar®, and in the United States

[0004] There is some evidence regarding certain new serotypes (e.g., 19A, etc.). “ O’Brien et al., 2004, Am J Epidemiol 159 :634-44”, “Whitney et al., 2003, N Engl J Med 348:1737-46”, “Kyaw et al., 2006, N Engl J Med 354:1455-63”, “Hicks et al., 2 007, J Infect Dis 196:1346-54”, “Traore e t al., 2009, Clin Infect Dis 48:S181-S18 9”. See also

[0005] Prevnar 13® is a 13-valent pneumococcal polysaccharide-protein conjugate vaccine containing serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9 V, 14, 18C, 19A, 19F, and 23F. For example, U.S. Patent Application Publication No. US2006 / 0 228380A1, “Prymula et al., 2006, Lancet 3 67:740-48” and “Kieninger et al., Safety an d Immunologic Non-inferiority of 13-vale nt Pneumococcal Conjugate Vaccine Compar ed to 7-valent Pneumococcal Conjugate Va ccine Given as a 4-Dose Series in Health y Infants and Toddlers, presented at the 48 th Annual ICAAC / ISDA 46 thAnnual Meet ing, Washington DC, October 25 - 28, 2008」 See also. Further, see 「Dagan et al., 1998, Infec t Immun. 66: 2093 - 2098」and 「Fattom, 1999, Vaccine 17:126」. However, in certain regions of the world there are limitations to the range of serotypes covered by Prevnar 13 (registered trademark), and in the United States there is some evidence regarding certain new serotypes (e.g., serotype 35B). 「O’Brien et al., 2004, Am J Epidemiol 15 9:634 - 44」, 「Whitney et al., 2003, N Engl J Med 348:1737 - 46」, 「Kyaw et al., 2006, N Engl J Med 354:1455 - 63」, 「Hicks et al., 2007, J Infect Dis 196:1346 - 54」, 「Traore et al., 2009, Clin Infect Dis 48:S181 - S1 89」, 「Olarte et al., 2017, J. Clin. Micro biology 55:724 - 734」.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non - Patent Documents

[0007]

Non - Patent Document 1

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0008] Current multivalent pneumococcal conjugate vaccines have been effective in reducing the incidence of pneumococcal diseases associated with the serotypes present in the vaccine. However, as described above, the prevalence of pneumococci expressing serotypes not present in the vaccine is increasing. Process conditions for new serotypes should be confirmed for each serotype with respect to conjugation efficiency. Certain serotypes (which include serotype 35B) present unique challenges due to their unique structures. Therefore, immunogenic serotype 35B multi- related pneumonia has an increased prevalence of pneumococci expressing serotypes not present in the vaccine. Process conditions for new serotypes should be confirmed for each serotype with respect to conjugation efficiency. Certain serotypes (which include serotype 35B) present unique challenges due to their unique structures. Therefore, immunogenic serotype 35B multi- related pneumonia related pneumonia There is a need for a saccharide-carrier protein conjugate and an improved process for preparing the same. is required.

Means for Solving the Problems

[0009] The present invention provides an immunogenic serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate and a process for preparing the same.

[0010] In one embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 7,000 kDa. is provided.

[0011] In another embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 7,000 kDa, wherein the conjugate contains lysine consumption of 3 mol / mol protein to 9 mol / mol protein. contains lysine consumption of 3 mol / mol protein to 9 mol / mol protein. is included.

[0012] In another embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 7,000 kDa, wherein the conjugate contains lysine consumption of 4 mol / mol carrier protein to 8 mol / mol protein. contains lysine consumption of 4 mol / mol carrier protein to 8 mol / mol protein. is included.

[0013] In one embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 5,000 kDa. is provided.

[0014] In another embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 5,000 kDa. Provide a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the conjugate contains a lysine consumption of 3 mol / mol protein to 9 mol / mol protein.

[0015] In another embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 5,000 kDa, wherein the conjugate contains a lysine consumption of 4 mol / mol carrier protein to 8 mol / mol protein.

[0016] In another embodiment, the present invention provides a composition comprising the conjugate, wherein the composition further comprises free polysaccharide less than 30% of the total polysaccharide amount and free protein less than 30% of the total protein amount.

[0017] In another embodiment, the present invention provides a composition comprising the conjugate, wherein the composition further comprises free polysaccharide less than 20% of the total polysaccharide amount and free protein less than 20% of the total protein amount.

[0018] In another aspect of the conjugate, the protein of the serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate is CRM197.

[0019] In another aspect of the composition, the protein of the polysaccharide-protein conjugate is CRM197.

[0020] In one embodiment, the present invention also provides a process for preparing the serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate, wherein the process activates the polysaccharide ​ includes causing activation, where the activation uses periodate within the range of 0.01 to 0.1 mole of periodate per mole of polysaccharide repeating unit. In another embodiment, the range of periodate is 0.03 to 0.06 mole of periodate per mole of polysaccharide repeating unit.

[0021] In another embodiment, the present invention provides a process for preparing the above immunogenic serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate, where the process includes conjugating the polysaccharide to the protein, where the conjugation is carried out at a conjugation temperature of 22°C to 38°C. In another embodiment, the conjugation temperature is 32°C to 36°C.

[0022] In another embodiment, the present invention provides a process for preparing the above serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate, where the process includes (i) causing activation of the polysaccharide (where the activation uses periodate within the range of 0.01 to 0 .1 mole of periodate per mole of polysaccharide repeating unit), and (ii) conjugating the polysaccharide to the protein (where the conjugation is carried out at a conjugation temperature of 22°C to 38°C).

[0023] In another embodiment, the present invention provides a process, where the activation of the polysaccharide uses periodate within the range of 0.03 to 0.06 mole of sodium metaperiodate per mole of polysaccharide repeating unit.

[0024] ​​​​​​In another embodiment, the present invention provides a process where the conjugation temperature is from 32°C to 36°C.

[0025] In another embodiment, the present invention provides a multivalent pneumococcal conjugate vaccine composition, wherein the composition contains the serotype 35 Streptococcus pneumoniae type B polysaccharide-protein conjugate provided in the above conjugate embodiments.

[0026] In another embodiment, the present invention provides a process for preparing the above serotype 35B Streptococcus pneumoniae polysaccharide-protein co njugate, wherein the polysaccharide is conjugated to the protein in an aprotic solvent. In a further embodiment, the aprotic solvent is DMSO. In another embodiment, the DMSO solvent contains less than 1.2% (v / v) water . In another embodiment, the DMSO solvent contains less than 0.6% (v / v) water . In another embodiment, the DMSO solvent contains less than 0.3% (v / v) water .

[0027] In another embodiment, the present invention provides a process for preparing the above serotype 35B Streptococcus pneumoniae polysaccharide-protein co njugate, wherein the conjugation of the polysaccharide to the protein is carried out in the presence of sodium chloride. In a further embodiment, the concentration of sodium chloride is about 5 - 15 mM.

Brief Description of the Drawings

[0028]

Figure 1

Mode for Carrying Out the Invention

[0029] The pneumococcal serotype 35B capsular polysaccharide contains an activation site within the polysaccharide chain backbone and is a complex molecule. During activation with periodate, acyclic triols are oxidized to reactive aldehydes, causing cleavage of the polysaccharide backbone. As a result, as the number of reactive aldehydes increases, the Mw of the polysaccharide decreases, limiting the effective activation range. The terminal aldehydes formed during activation further limit the degree of polysaccharide cross-linking with the carrier protein, resulting in low Mw conjugates.

[0030] Due to the complexity of this structure, efforts to conjugate serotype 35B polysaccharide have met with limited success when using conventional activation and conjugation processes. For example, when activating serotype 35B polysaccharide with a standard range of periodate used for polysaccharides from other pneumococcal serotypes, conjugates that are too small are obtained. Furthermore, typical Ps:Pr (ratio of polysaccharide to carrier protein), polysaccharide concentration, carrier protein concentration, and temperature ranges used in the conjugation reaction have resulted in inadequate conjugate attributes such as low conjugate Mw, low lysine consumption, and high levels of free Ps and carrier protein.

[0031] Regarding the activation step for generating immunogenic serotype 35B polysaccharide-protein conjugates, a preferred range of periodate was identified. When activating below the preferred periodate range, polysaccharide-protein conjugates with the desired size were obtained, Due to the lack of aldehydes available for conjugation, insufficient conjugate attributes such as low lysine consumption, high free protein and high free polysaccharides were brought about. When activated beyond the preferred periodate range, low Mw conjugates with low immunogenic potential were generated due to the degree of polysaccharide size reduction during the activation reaction .

[0032] Furthermore, it was confirmed that Streptococcus pneumoniae serotype 35B polysaccharide is sensitive to the water content during the conjugation reaction and that the conjugation reaction can be inhibited . Water interferes with the serotype 35B polysaccharide conjugation reaction by promoting the aggregation of proteins and polysaccharides. In an organic conjugation reaction, water can be introduced when adding additional components such as salts and reducing agents to the reaction. The present invention disclosed herein provides a method for reducing the water content during the conjugation reaction by eliminating weak reducing agents and including sodium chloride in the pre-lyophilized formulation . Furthermore, the pre-lyophilized formulations of proteins and polysaccharides were incorporated together into a co-lyophilized formulation. This eliminated the mixing of polysaccharides and proteins, initialized conjugation immediately after dissolution, reduced the absorption of moisture in the air, and further limited the water content during the reaction . . . . . . . .

[0033] A preferred temperature range for the Streptococcus pneumoniae serotype 35B polysaccharide conjugation reaction that produces an immunogenic serotype 35B polysaccharide-protein conjugate with improved conjugate attributes was identified. Conjugation at temperatures lower than this preferred range . . In Yon, due to the decrease in reaction rate, small conjugates with insufficient conjugate attributes (especially with a large amount of free Ps) were produced. At temperatures exceeding the preferred temperature range for the conjugation reaction, the stability of the protein became insufficient, and the protein might aggregate. For the conjugation reaction, at temperatures exceeding the preferred temperature range, the stability of the protein became insufficient, and the protein might aggregate. For the conjugation reaction, at temperatures exceeding the preferred temperature range, the stability of the protein became insufficient, and the protein might aggregate. For the conjugation reaction, at temperatures exceeding the preferred temperature range, the stability of the protein became insufficient, and the protein might aggregate.

[0034] As used herein, the term "carrier protein" refers to DT (diphtheria toxoid), TT (tetanus toxoid), or CRM197. "Carrier protein" is also referred to as "protein". In a preferred embodiment, "carrier protein" means CRM197. As used herein, the term "carrier protein" refers to DT (diphtheria toxoid), TT (tetanus toxoid), or CRM197. "Carrier protein" is also referred to as "protein". In a preferred embodiment, "carrier protein" means CRM197. As used herein, the term "carrier protein" refers to DT (diphtheria toxoid), TT (tetanus toxoid), or CRM197. "Carrier protein" is also referred to as "protein". In a preferred embodiment, "carrier protein" means CRM197. As used herein, the term "carrier protein" refers to DT (diphtheria toxoid), TT (tetanus toxoid), or CRM197. "Carrier protein" is also referred to as "protein". In a preferred embodiment, "carrier protein" means CRM197.

[0035] As used herein, the term "free protein" means a protein that is present in the composition but not covalently bound to a polysaccharide. The term "conjugated protein" means a protein covalently bound to a polysaccharide. The term "total protein" means all the proteins present in the composition, including free proteins and conjugated proteins. As used herein, the term "free protein" means a protein that is present in the composition but not covalently bound to a polysaccharide. The term "conjugated protein" means a protein covalently bound to a polysaccharide. The term "total protein" means all the proteins present in the composition, including free proteins and conjugated proteins. As used herein, the term "free protein" means a protein that is present in the composition but not covalently bound to a polysaccharide. The term "conjugated protein" means a protein covalently bound to a polysaccharide. The term "total protein" means all the proteins present in the composition, including free proteins and conjugated proteins. As used herein, the term "free protein" means a protein that is present in the composition but not covalently bound to a polysaccharide. The term "conjugated protein" means a protein covalently bound to a polysaccharide. The term "total protein" means all the proteins present in the composition, including free proteins and conjugated proteins. As used herein, the term "free protein" means a protein that is present in the composition but not covalently bound to a polysaccharide. The term "conjugated protein" means a protein covalently bound to a polysaccharide. The term "total protein" means all the proteins present in the composition, including free proteins and conjugated proteins.

[0036] As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides. As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides. As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides. As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides. As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides. As used herein, the term "polysaccharide" (Ps) refers to pneumococcal capsular polysaccharide. The term "free polysaccharide" means a polysaccharide that is present in the composition but not covalently bound to a carrier protein. The term "conjugated polysaccharide" means a polysaccharide covalently bound to a protein. The term "total polysaccharide" means all the polysaccharides present in the composition, including free polysaccharides and conjugated polysaccharides.

[0037] As used herein, "periodate" includes both periodate and periodic acid. This term further includes both metaperiodate (IO4-) and ortho periodate (IO6-), as well as various salts of periodate (e.g., sodium periodate and potassium periodate). In a preferred embodiment, "periodate" refers to sodium metaperiodate.

[0038] As used herein, the term "Mw" refers to the weight average molecular weight and is typically expressed in Da or kDa. Mw takes into account that larger molecules contribute more to the total mass of a polymer sample. Mw can be measured by techniques such as static

[0039] light scattering, small angle neutron scattering, X-ray scattering and sedimentation velocity. As used herein, the term "Mn" refers to the number average molecular weight and is typically expressed in Da or kDa. Mn is calculated by dividing the total weight of the sample by the number of molecules in the sample and can be measured by techniques such as gel

[0040] permeation chromatography, viscosity measurements (by the Mark- Houwink equation), colligative methods (e.g., vapor pressure osmometry), end group quantification or proton NMR. Mw / Mn Pr can be measured by HPSEC / UV / MALS / RI assay. In the present invention, in one embodiment, the Ps:Pr ratio for a serotype 35B polysaccharide-protein conjugate is in the range of 0.5 to 2.0.

[0041] As used herein, the term "comprise" when used in connection with the immunogenic compositions and / or pneumococcal conjugate vaccines of the present invention indicates the inclusion of any other optional components such as adjuvants and excipients (subject to the limitation of the term "consisting of" relating to antigen mixtures). The term "consisting of" when used in connection with a mixture of polysaccharide-protein conjugates indicates a mixture that has those specific pneumococcal polysaccharide-protein conjugates but does not have other pneumococcal polysaccharide-protein conjugates from different serotypes. As used herein, the term "activation step" refers to the process of reacting adjacent diols on

[0042] serotype 35B pneumococcal polysaccharide with an oxidizing agent to form a reactive aldehyde. As used herein, the term "conjugation step" refers to the process of conjugating a reactive aldehyde on serotype 35B pneumococcal polysaccharide to a lysine group on a carrier protein

[0043] using reductive amination. Unless otherwise specified, all ranges described herein include the lower and upper limits described.

[0044]

[0045] ​​ Definitions and Abbreviations As used throughout this specification and the appended claims, the following abbreviations apply:

Table 1

[0046] General Methods for Preparing Polyvalent Pneumococcal Polysaccharide Conjugate Vaccines Capsular Polysaccharide Bacterial capsular polysaccharides, particularly those that have been used as antigens, are suitable for use in the present invention and can be readily identified by methods for discriminating immunogenic and / or antigenic polysaccharides. Examples of bacterial capsular polysaccharides derived from Streptococcus pneumoniae are the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18 C, 19A, 19F, 20 (20A and 20B), 22F, 23A, 23B, 23F, 2 4F, 33F, 35B, 35F or 38.

[0047]

[0047] Polysaccharides can be purified by known techniques. The present invention is not limited to polysaccharides purified from natural sources, but polysaccharides can be obtained by other methods such as total synthesis or partial synthesis. Capsular polysaccharides derived from Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be separated from bacteria by known methods and can be sized to some extent (see, for example, European Patent Nos. EP 497524 and EP497525); and preferably, by microfluidization achieved using a homogenizer or by chemical hydrolysis, 497524 and EP497525); and preferably, by microfluidization achieved using a homogenizer or by chemical hydrolysis, It can be separated and sized. Streptococcus pneumoniae strains corresponding to each polysaccharide serotype can be grown in a Todd-Hewitt-based medium. Subsequently, the individual polysaccharides can be purified through standard steps such as centrifugation, precipitation, and ultrafiltration. See, for example, U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. The polysaccharides can be sized to lower viscosity and / or improve the consistency between batches of the conjugated product. The purified polysaccharides can be chemically activated using standard techniques to introduce functionality capable of reacting with a carrier protein. Chemical activation of the polysaccharides and subsequent conjugation to the carrier protein are achieved by the methods described in U.S. Patent Nos. 4,365,170, 4,673,574, and 4,902,506. Briefly, the pneumococcal polysaccharide is reacted with a periodate-based oxidizing agent (e.g., sodium periodate, potassium periodate, or periodic acid), thereby causing oxidative cleavage of adjacent hydroxyl groups to generate reactive aldehyde groups. The appropriate molar equivalent of the periodate salt (e.g., sodium periodate, sodium metaperiodate, etc.) includes 0.05 to 0.5 molar equivalents (the molar ratio of periodate to the repeating unit of the polysaccharide) or 0.1 to 0.5 molar equivalents. The reaction of the periodate can vary from 30 minutes to 24 hours depending on the conformation of the diol (e.g., acyclic diol, cis-diol, trans-diol) that controls the accessibility of the reactive hydroxyl groups to sodium periodate.

[0048] ​​​​​​​​​​​​​​​​​

[0049] The term "periodate" includes both periodates and periodic acids ; this term includes both metaperiodates (IO 4- ) and orthoperiodates (I O 6- ), and also includes various salts of periodates (e.g., sodium periodate and potassium periodate). The capsular polysaccharide can be oxidized in the presence of metaperiodate or in the presence of sodium periodate (NaIO 4 4 ). Further , the capsular polysaccharide can be oxidized in the presence of orthoperiodate or in the presence of periodic acid .

[0050] The purified polysaccharide can also be linked to a linker. Each capsular polysaccharide, once activated or linked to a linker, can be separately conjugated to a carrier protein to form a conjugate polysaccharide. The polysaccharide conjugate can be prepared by known coupling techniques .

[0051] The polysaccharide can be coupled to a linker to form a polysaccharide-linker intermediate in which the free end of the linker is an ester group . Thus, the linker is a linker having at least one end that is an ester group. The other end is selected so that it can react with the polysaccharide to form a polysaccharide-linker intermediate

[0052] The polysaccharide can be coupled to the linker using a primary amine group within the polysaccharide . In this case, the linker typically has ester groups at both ends ​This allows one of the ester groups to be attached to the primary acyl group of the polysaccharide by a nucleophilic acyl substitution reaction. By reacting the amine group with the polysaccharide, coupling can be performed. This results in a polysaccharide-linker intermediate in which is coupled to the linker via an amide bond. Thus, the linker contains a first ester group for reacting with a primary amine group in the polysaccharide. and a bifunctional linker that provides a second ester group for reaction with a primary amine group in the carrier molecule. A typical linker is adipic acid N-hydroxysuccinimide diester. The name is SIDEA.

[0053] The coupling can also be performed indirectly, i.e., by coupling to a linker. This can also be accomplished with an additional linker that is used to derivatize the polysaccharide prior to glycosylating. It is possible.

[0054] Polysaccharides are fused to additional linkers using the carbonyl group at the reducing end of the polysaccharide. This coupling involves two steps: a1) reacting a carbonyl group with an additional linker; and (a2) reacting said additional linker. In these embodiments, the additional linker may be reacted with the free end of the linker. Typically, the amine has a primary amine group at each end, thereby One of the amine groups is reacted with a carbonyl group in the polysaccharide by reductive amination, (a1) can be carried out by reacting a primary aryl group with a carbonyl group in the polysaccharide. A hydrazide or hydroxylamino group is suitable. The amine group typically has an additional functional group that allows for the possibility of polysaccharide (Ps)-Ps coupling. It is present at both ends of the linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via a C-N bond. The polysaccharide can be coupled to the additional linker using different groups (especially carboxyl groups) within the polysaccharide. This coupling involves the following two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the said linker. In this case, the additional linker typically has primary amine groups at both ends, so that one of the primary amine groups can be reacted with the carboxyl group within the polysaccharide by EDAC activation to enable step (a1). A primary amine group capable of reacting with the EDAC-activated carboxyl group within the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond.

[0055] The polysaccharide can be coupled to the additional linker using different groups (especially carboxyl groups) within the polysaccharide. This coupling involves the following two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the said linker. In this case, the additional linker typically has primary amine groups at both ends, so that one of the primary amine groups can be reacted with the carboxyl group within the polysaccharide by EDAC activation to enable step (a1). A primary amine group capable of reacting with the EDAC-activated carboxyl group within the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond. It is present at both ends of the linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via a C-N bond. The polysaccharide can be coupled to the additional linker using different groups (especially carboxyl groups) within the polysaccharide. This coupling involves the following two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the said linker. In this case, the additional linker typically has primary amine groups at both ends, so that one of the primary amine groups can be reacted with the carboxyl group within the polysaccharide by EDAC activation to enable step (a1). A primary amine group capable of reacting with the EDAC-activated carboxyl group within the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond. The polysaccharide can be coupled to the additional linker using different groups (especially carboxyl groups) within the polysaccharide. This coupling involves the following two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the said linker. In this case, the additional linker typically has primary amine groups at both ends, so that one of the primary amine groups can be reacted with the carboxyl group within the polysaccharide by EDAC activation to enable step (a1). A primary amine group capable of reacting with the EDAC-activated carboxyl group within the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond. The polysaccharide can be coupled to the additional linker using different groups (especially carboxyl groups) within the polysaccharide. This coupling involves the following two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the said linker. In this case, the additional linker typically has primary amine groups at both ends, so that one of the primary amine groups can be reacted with the carboxyl group within the polysaccharide by EDAC activation to enable step (a1). A primary amine group capable of reacting with the EDAC-activated carboxyl group within the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. This reaction results in a polysaccharide-added linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond.

[0056] Carrier Protein In a particular embodiment of the present invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic mutant (i.e., toxoid) of diphtheria toxin. CRM197 can be isolated from a culture of Corynebacterium diphtheria C7 strain (β197) grown in a medium based on casamino acids and yeast extract. Further, CRM197 is described in U.S. Patent No. 5,614,382. CRM197 is a non-toxic mutant (i.e., toxoid) of diphtheria toxin. CRM197 can be isolated from a culture of Corynebacterium diphtheria C7 strain (β197) grown in a medium based on casamino acids and yeast extract. It can be isolated from a culture of Corynebacterium diphtheria C7 strain (β197) grown in a medium based on casamino acids and yeast extract. Further, CRM197 is described in U.S. Patent No. 5,614,382. It can be recombinantly prepared according to the method. Typically, CRM197 is purified by a combination of ultrafiltration and ammonium sulfate precipitation and ion exchange chromatography. In some embodiments, CRM197 is prepared in Pseudomonas fluorescens using Pfenex Expression Technology TM (Pfenex Inc., San Diego, CA). Another suitable carrier protein includes additional inactivated bacterial toxins, such as DT (diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, pertussis toxin, cholera toxin (e.g., as described in

[0057] International Patent Application Publication No. WO2004 / 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 Publication No. WO02 / 091998), pneumococcal surface adhesin protein (PsaA), C5a peptidase derived from Streptococcus group A or group B, or protein D of Haemophilus influenzae, pneumococcal neumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13) [which is ply detoxified in some way, such as dP ... ... ... ... ... LY-GMBS (see International Patent Application Publication No. WO04 / 081515) or d containing PLY-formol), PhtX (e.g., PhtA, PhtB, PhtD , PhtE, and fusions of Pht proteins, e.g., PhtDE fusion, PhtBE fusion) (see International Patent Application Publication Nos. WO01 / 98334 and WO03 / 54007 ) can also be used. Another protein, e.g., ovalbumin , keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (derived from N. meningitidis (N .meningitidis)), PD (Haemophilus influenzae protein D; e.g., see European Patent No. EP0594610B ), or immunologically functional equivalents thereof, synthetic peptides (see European Patent Nos. E P0378881 and EP0427347), heat shock proteins (see International Patent Application Publication Nos. WO93 / 17712 and WO94 / 03208 ), pertussis proteins (see International Patent Application Publication No. WO98 / 58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (see International Patent Application Publication No. WO91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes from antigens derived from various pathogens ("Falugi et al., 2001, Eur J Immunol 31:3816-3824"), e.g., N19 protein ("Ba raldoi et al., 2004, Infect Immun 72:488 ), etc. can also be used. Another protein, e.g., ovalbumin raldoi et al., 2004, Infect Immun 72:488 (see also "4-7"), iron uptake proteins (see International Patent Application Publication No. WO01 / 72 337), toxin A or toxin B of C. difficile (see International Patent Publication No. WO00 / 61761), and flagellin (see "Ben-Yedidia et al., 1998, Immunol Lett 64:9") can also be used as carrier proteins .

[0058] When using a multivalent vaccine, the second carrier can be used for one or more antigens in the multivalent vaccine. The second carrier protein is preferably a non-toxic and non-reactogenic protein that is available in sufficient quantity and purity. The second carrier protein is further conjugated or bound to an antigen (e.g., pneumococcal polysaccharide) to enhance the immunogenicity of the antigen. The carrier protein should follow standard conjugation procedures. Each capsular polysaccharide that is not conjugated to the first carrier protein can be conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule is conjugated to a single carrier protein ). Capsular polysaccharides that are not conjugated to the first carrier protein can be conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to a single carrier protein ). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein. Another DT variant, for example, the following ones can be used as the second carrier protein: CRM176, CRM22 ). ). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein. Another DT variant, for example, the following ones can be used as the second carrier protein: CRM176, CRM22 ​ 8. CRM45 (Uchida et al., 1973, J Biol Chem 218:3838 - 3844); CRM9, CRM45, CRM102, CRM103 and CRM107, and another variant described by Nicholls and Youle in "Geneticall y Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992"; deletion of Glu - 148 or mutation to Asp, Gln or Ser, and / or deletion of Ala 158 or mutation to Gly, and another mutation disclosed in U.S. Patent No. 4,709,017 or U.S. Pat ent No. 4,950,740; mutation of at least one of the residues Lys 516, Lys 526, Phe 530 and / or Lys 534 , and another mutation disclosed in U.S. Patent No. 5,917,017 or U.S. Patent No. 6,455,673 ; or a fragment disclosed in U.S. Patent No. 5,843,711 .

[0059] Conjugation by Reductive Amination The covalent attachment of the polysaccharide to the carrier protein is carried out via reductive amination which directly couples an amine - reactive moiety on the polysaccharide to the primary amine groups (mainly lysine residues) of the protein. As is well known, the reductive amination reaction proceeds via a two - step mechanism . First, the aldehyde group (R - CHO) of molecule 1 is reacted with the primary amine group (R’ - NH2) of molecule 2 to form a Schiff base intermediate represented by the formula R - CH=N - R’. In the second step, the Schiff base is reduced to form a compound represented by the formula R - CH2 - NH - R’ To form the resulting amino compound. Although many reducing agents can be used, in most cases, Sodium cyanoborohydride (NaCNBH 3 ) and other highly selective reducing agents are used . This is because such reagents specifically reduce only the imine functional group of the Schiff base .

[0060] All polysaccharides have an aldehyde functional group (terminal aldehyde functional group) at the end of the chain . Conjugation methods involving reductive amination of polysaccharides are very generally applicable . And when there are no other aldehyde functional groups (intra-chain aldehyde functional groups) within the repeating unit, such methods can yield conjugates in which the polysaccharide molecule is coupled to a single molecule of the carrier protein .

[0061] Typical reducing agents are cyanoborohydride salts such as sodium cyanoborohydride . The imine-selective reducing agent commonly used is sodium cyanoborohydride, but it is also possible to use other cyanoborohydride salts including potassium cyanoborohydride . Due to the difference in the starting cyanide level in the sodium cyanoborohydride reagent lot and the residual cyanide in the conjugation reaction, there may be a lack of consistency in the conjugation performance, and as a result, product attributes such as the conjugation size and the conjugate Ps to CR M197 ratio may vary. By controlling and / or reducing the free cyanide level in the final reaction product, the variability of the conjugation can be reduced . .

[0062] Residual unreacted aldehydes on the polysaccharide may, in some cases, be strong reducing agents such as sodium borohydride ​Reduce by adding the original agent. Generally, the use of a strong reducing agent is preferred. However, For some polysaccharides, it is preferred to avoid this step. For example, Streptococcus pneumoniae serotype 5 contains a ketone group that can easily react with a strong reducing agent. In this case, it is preferred to avoid the reduction step to protect the antigen structure of the polysaccharide.

[0063] Following conjugation, the polysaccharide-protein conjugate is purified by any technique well known to those skilled in the art (which includes concentration / diafiltration operations, ultrafiltration, precipitation / elution, column chromatography and depth filtration) to remove excess conjugation reagent and residual free protein and free polysaccharide. See, for example, U.S. Patent No. 6,146,902. In one embodiment, the purification step is by ultrafiltration.

[0064] In one embodiment, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 500 kDa to 10,000 kDa or 1000 kDa to 10,000 kDa or 1,000 kDa to 9,000 kDa or 1,000 kDa to 8 ,000 kDa or 1,000 kDa to 7,000 kDa or 1,000 kDa to 6,0 00 kDa. Preferably, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 7,000 kDa. Further more preferably, the present invention provides a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate having a molecular weight of 1,000 kDa to 5,000 kDa.

[0065] ​​​​In one embodiment, the present invention provides a process for preparing a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate as described in the conjugate embodiments above, where the process includes activating the polysaccharide, where the activation uses periodate within the range of 0.01 to 0.1 mole of periodate per mole of polysaccharide repeating unit. In another embodiment, the range of periodate is 0.03 to 0.06 mole of periodate per mole of polysaccharide repeating unit. Process for preparing a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate which includes activating the polysaccharide, where the activation uses periodate within the range of 0.01 to 0.1 mole of periodate per mole of polysaccharide repeating unit. In another embodiment, the range of periodate is 0.03 to 0.06 mole of periodate per mole of polysaccharide repeating unit.

[0066] In another embodiment, the present invention provides a process for preparing an immunogenic serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate as described in the conjugate embodiments above, where the process includes conjugating the polysaccharide to the protein, where the conjugation is carried out at a conjugation temperature of 22°C to 38°C. In another embodiment, the conjugation temperature is 32°C to 36°C. Process for preparing an immunogenic serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate which includes conjugating the polysaccharide to the protein, where the conjugation is carried out at a conjugation temperature of 22°C to 38°C. In another embodiment, the conjugation temperature is 32°C to 36°C.

[0067] In another embodiment, the present invention provides a process for preparing a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate as described in the conjugate embodiments above, where the process includes activating the polysaccharide (where the activation uses periodate within the range of 0.01 to 0.1 mole of periodate per mole of polysaccharide repeating unit) and conjugating the polysaccharide to the protein (where the conjugation is carried out at a conjugation temperature of 22°C to 38°C). Process for preparing a serotype 35B Streptococcus pneumoniae polysaccharide-protein conjugate which includes activating the polysaccharide (where the activation uses periodate within the range of 0.01 to 0.1 mole of periodate per mole of polysaccharide repeating unit) and conjugating the polysaccharide to the protein (where the conjugation is carried out at a conjugation temperature of 22°C to 38°C).

[0068] ​​In another embodiment, the present invention provides a process, wherein the activation of the polysaccharide is carried out on the polysaccharide using periodate within the range of 0.03 to 0.06 moles of sodium metaperiodate per mole of the polysaccharide repeating unit.

[0069] In another embodiment, the present invention provides a process, wherein the conjugation temperature is 32°C to 36°C.

[0070] In one embodiment of the present invention, an aprotic solvent is used in the conjugation reaction. In another embodiment of the present invention, DMSO (dimethyl sulfoxide) is used as the aprotic solvent in the conjugation reaction.

[0071] In another embodiment, the conjugation is carried out in a DMSO solvent containing sodium chloride. In another embodiment, the sodium chloride concentration is about 1 mM to 50 mM. In another embodiment, the sodium chloride concentration is about 5 mM to 15 mM.

[0072] In another embodiment, the conjugation is carried out in a DMSO solvent containing less than 1.2% water (v / v). In another embodiment, the conjugation is carried out in a DMSO solvent containing less than 0.6% water (v / v). In another embodiment, the conjugation is carried out in a DMSO solvent containing less than 0.3% water (v / v).

[0073] In one embodiment, the conjugation is carried out in a DMSO solvent using an activated polysaccharide having an aldehyde per repeating unit within the range of 0.01 to 0.1. In another embodiment, the conjugation is carried out in a DMSO solvent using an activated polysaccharide having an aldehyde per repeating unit within the range of 0.03 to 0.06. ​​​It is carried out in a DMSO solvent using an activated polysaccharide having rhamnaldehyde.

[0074] In one embodiment, the conjugation is carried out in a DMSO solvent using an activated polysaccharide having a molecular weight in the range of 30 to 200 kDa. In another embodiment, the conjugation is carried out in a DMSO solvent using an activated polysaccharide having a molecular weight in the range of 40 to 100 kDa. In another embodiment, the conjugation reaction is carried out using a polysaccharide of reduced size prepared through activation with periodate.

[0075] In another embodiment, the immunogenic composition is conjugated to one or more carrier proteins and contains a capsular polysaccharide derived from at least one of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20 (20A or 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F or 38. Preferably, the saccharides derived from specific serotypes are not conjugated to two or more carrier proteins.

[0076] Polyvalent Polysaccharide-Protein Conjugate Vaccine After purifying the individual glycoconjugates, they are formulated to prepare the immunogenic composition of the present invention. These pneumococcal conjugates are prepared by separate processes and bulk formulated into a single dosage form.

[0077]

[0078] Pharmaceutical / Vaccine Composition The present invention further relates to a pharma- ceutical comprising any of the above-described polysaccharide serotype combinations. or consisting essentially of, together with suitable carriers and adjuvants; and compositions (including pharmaceutical compositions, immunogenic compositions and vaccine compositions) consisting of them. The composition includes 2 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 5, 26, 27, 28, 29, 30, 31, 32, 33, 34 or up to 35 different polysaccharides It may comprise or consist essentially of a catecholamine-protein conjugate. and, wherein each of the conjugates The antibody is a heterologous antibody conjugated to either a first carrier protein or a second carrier protein. and wherein the capsular polysaccharides are selected from the group consisting of serotypes 1, 2, 3, and 4 of Streptococcus pneumoniae. , 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14 , 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20 (20A or 20B), 22F, 23A, 23B, 23F, 24F, 33F, 35B, 35F or 3 Capsular polysaccharides from at least one of the 8 species are conjugated to CRM197.

[0079] In one embodiment, the present invention relates to a serotype 3 conjugate as provided in the above conjugate embodiment. Polyvalent pneumococcal conjugates containing 5B pneumococcal polysaccharide-protein conjugates Provide the DUGATE vaccine.

[0080] Formulation of the polysaccharide-protein conjugates may be performed using art-recognized methods. It can be achieved by using. For example, each pneumococcal conjugate 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. In a preferred embodiment, the vaccine composition is formulated in an L - histidine buffer containing sodium chloride. Examples of such vehicles include, but are not limited to: (1) Aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59

[0081] As defined herein, an "adjuvant" is a substance that helps enhance the immunogenicity of the immunogenic composition of the present invention. An immunoadjuvant can enhance the immune response to an antigen that is weakly immunogenic when administered alone (e.g., does not induce or induces a weak antibody titer or cellular immune response), can increase the antibody titer against the antigen, and / or can reduce the dose of the antigen effective to achieve an immune response in an individual. Thus, adjuvants are often given to enhance the immune response and are well known to those skilled in the art. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to, the following: (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59

[0082] As defined herein, an "adjuvant" is a substance that helps enhance the immunogenicity of the immunogenic composition of the present invention. An immunoadjuvant can enhance the immune response to an antigen that is weakly immunogenic when administered alone (e.g., does not induce or induces a weak antibody titer or cellular immune response), can increase the antibody titer against the antigen, and / or can reduce the dose of the antigen effective to achieve an immune response in an individual. Thus, adjuvants are often given to enhance the immune response and are well known to those skilled in the art. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to, the following: (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 can be mentioned: (1) Aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (2) Water - in - oil emulsion formulations (in the presence or absence of other specific immunostimulants such as muramyl peptides (defined below) or bacterial cell wall components), for example, (a) MF59 (International Patent Application Publication No. WO90 / 14837) [This contains 5% squalene, 0.5% T ween80 and 0.5% Span 85 (optionally containing various amounts of M TP-PE), and is formulated into submicron particles using a microfluidizer (e.g., Model 110Y m icrofluidizer (Microfluidics, Newton, MA)]; (b) SAF [This contains 10% squalene, 0 .4% Tween80, 5% pluronic block polymer L121 and thr-MDP , and is microfluidized into a submicron emulsion or vortexed to produce a large-particle-size emulsion]; (c) Ribi adjuvant system (RAS) (Corixa, Hamilton, MT) [This contains 2% squ alene, 0.2% Tween80, and one or more bacterial cell wall components selected from the group consisting of 3-O-deacylated monophosphoryl TM lipid A (MPL )(described in U.S. Patent No. 4,912,094), trehalose dimycolate (TDM), and cell wall skeleton (CWS) (preferably, MPL + CWS (Detox )); and, (d) Montanide ISA; ); and, (d) Montanide ISA; TM )(described in U.S. Patent No. 4,912,094), trehalose dimycolate (TDM), and cell wall skeleton (CWS) (preferably, MPL + CWS (Detox ))]; and, (d) Montanide ISA; ); and, (d) Montanide ISA; TM ))]; and, (d) Montanide ISA; ); and, (d) Montanide ISA; (3) Saponin adjuvants [e.g., Quil A or STIMULON TM Q S-21 (Antigenics, Framingham, MA) (see, e.g., U.S. Pat ent No. 5,057,540) may be used, or particles generated from the adjuvant, e.g., ISCOM (cholesterol, saponin, phospholipid, and amphiphilic ); and, (d) Montanide ISA; (An immunopotentiating complex formed by a combination of proteins), and Iscomatri x (registered trademark) (which has essentially the same structure as ISCOM but does not contain proteins )); (4) Bacterial lipopolysaccharides, synthetic lipid A analogs [e.g., aminoalkyl glucosamine phosph ate compounds (AGP), or derivatives or analogs thereof (which are available from Corixa and are described in U.S. Patent No. 6,113,918); an example of such an AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylam ino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoy loxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecano ylamino}-β-D-glucopyranoside (which is also known as 529 (previously known as RC529), formulated as an aqueous form or a stable emulsion )); (5) Synthetic polynucleotides [e.g., oligonucleotides containing one or more CpG motifs (U.S. Patent No. 6,207,646)]; (6) Cytokines [e.g., interleukins (e.g., IL-1, IL-2, IL -4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), inter ferons (e.g., γ 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 [e.g., trimers of complement component C3d]. );

[0083] ​In another embodiment, the adjuvant is two, three or more of the above adjuvants a mixture, for example, SBAS2 (water-in-oil emulsion containing 3-deacylated monophosphoryl lipid A and QS21 as well).

[0084] Examples of muramyl peptides include, but are not limited to, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L -alanine-2-(1’-2’ dipalmitoyl-sn-glycero-3-hydroxyphospho ryloxy)-ethylamine (MTP-PE), and the like.

[0085] In a particular embodiment, the adjuvant is an aluminum salt. The aluminum salt adjuvant can be a alum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in "Harl ow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Lab oratory)" and "Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-49 3)". Examples of the aluminum salt include, but are not limited to, hydrated al umina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, aluminum hydroxide gel, Superfos, Amphogel, aluminum hydroxide (III), aluminum hydroxyphosphate sulfate (aluminum phosphate adjuvant (APA), amorphous alumina, aluminum trihydroxide, or trihydroxyaluminum, etc. can be mentioned.

[0086] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is prepared by mixing aluminum chloride and sodium phosphate in a volume ratio of 1:1 to precipitate aluminum hydroxyphosphate. After the mixing process, the substance is size-reduced using a high-shear mixer to achieve a monodisperse particle size distribution. Then, the product is diafiltered against physiological saline and sterilized (steam sterilization or autoclaving).

[0087] Commercially available Al(OH) 3 (e.g., "Denmark / Accurate Chemical and Scientific Co., Westbury, NY" 's Alhydrogel or Superfos) can be used to adsorb proteins. Protein adsorption depends on the pI (isoelectric pH) of the protein and the pH of the medium in another embodiment. Proteins with a low pI adsorb more strongly to positively charged aluminum ions than proteins with a high pI. Aluminum salts can construct a reservoir of antigens that are slowly released over a period of 2 - 3 weeks, be involved in the non-specific activation of macrophages and complement activation, and / or stimulate the innate immune system (presumably through the stimulation of uric acid). See, for example, "Lambrecht et al., 2009, Curr Opin Immunol 21:23".

[0088] The monovalent bulk aqueous conjugate is typically mixed together, and all except 6B Dilute to the target of 8 μg / mL for that serotype and dilute 6B to the target of 16 μg / mL. After dilution, filter-sterilize the batch and aseptically add an equal volume of aluminum phosphate adjuvant to achieve a final aluminum concentration of 250 μg / mL. After the adjuvant is added, the formulated batch is placed into disposable 0.5 mL / dose vials.

[0089] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, such as a CpG-containing oligonucleotide, particularly a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, which is obtainable from the "Coley Pharmaceutical Group".

[0090] The terms "CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide" and similar terms refer to nucleotide molecules of 6-50 nucleotides in length that contain an unmethylated CpG moiety. See, for example, "Wang et al., 2003, Vaccine 21:4297". In another embodiment, any other definition recognized in the art for such terms is intended. CpG-containing oligonucleotides include oligonucleotides modified with any synthetic internucleoside linkage, modified bases and / or modified sugars.

[0091] Methods of using CpG oligonucleotides are well known in the art and are described, for example, in "Sur et al., 1999, J Immunol. 162:6" ​​​​​​​​​​​​​​"284-93", "Verthelyi, 2006, Methods Mol Me d. 127:139-58" and "Yasuda et al., 2006, Cr it Rev Ther Drug Carrier Syst. 23:89-110 is described in."

[0092] Administration / Dosage The compositions and formulations of the present invention are used to protect or treat humans who are susceptible to infection (e.g., pneumococcal infection) by administering the vaccine via a systemic or mucosal route Thereby, it can be used. In one embodiment, the present invention provides a method for inducing an immune response against pneumococcal capsular polysaccharide conjugate Here, the method includes administering an immunologically effective amount of the immunogenic composition of the present invention to a human. In another embodiment, the present invention provides a method for vaccinating a human against pneumococcal infection, where the method includes the step of administering an immunologically effective amount of the immunogenic composition of the present invention to a human." The optimal amount of the components for a specific vaccine can be confirmed by standard tests including observing an appropriate immune response in the subject

[0093] For example, in another embodiment, the dosage for vaccination of humans is determined by extrapolation from animal tests to human data Another embodiment, the dosage is determined empirically." The "effective amount" of the composition of the present invention refers to the dosage required to induce antibodies that significantly reduce the infectivity or severity of a microorganism (e.g., pneumococcus coccus) during subsequent challenge."

[0094]

[0095] ​​​The method of the present invention can be used for the prevention and / or reduction of major clinical syndromes caused by microorganisms (e.g., Streptococcus pneumoniae), which include both invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis). Clinical syndromes (including both invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis)). It can be used for the prevention and / or reduction of major clinical syndromes caused by microorganisms (e.g., Streptococcus pneumoniae), which include both invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis). It can be used for the prevention and / or reduction of major clinical syndromes caused by microorganisms (e.g., Streptococcus pneumoniae), which include both invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

[0096] Administration of the composition of the present invention may include one or more of the following: injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous route; or via mucosal administration to the oral / dietary, respiratory, or urogenital tract. In one embodiment, intranasal administration is used for treating pneumonia or otitis media (because it can more effectively suppress pneumococcal nasopharyngeal carriage, thereby attenuating the infection at its initial stage). Administration of the composition of the present invention may include one or more of the following: injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous route; or via mucosal administration to the oral / dietary, respiratory, or urogenital tract. In one embodiment, intranasal administration is used for treating pneumonia or otitis media (because it can more effectively suppress pneumococcal nasopharyngeal carriage, thereby attenuating the infection at its initial stage). Administration of the composition of the present invention may include one or more of the following: injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous route; or via mucosal administration to the oral / dietary, respiratory, or urogenital tract. In one embodiment, intranasal administration is used for treating pneumonia or otitis media (because it can more effectively suppress pneumococcal nasopharyngeal carriage, thereby attenuating the infection at its initial stage). Administration of the composition of the present invention may include one or more of the following: injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous route; or via mucosal administration to the oral / dietary, respiratory, or urogenital tract. In one embodiment, intranasal administration is used for treating pneumonia or otitis media (because it can more effectively suppress pneumococcal nasopharyngeal carriage, thereby attenuating the infection at its initial stage). Administration of the composition of the present invention may include one or more of the following: injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous route; or via mucosal administration to the oral / dietary, respiratory, or urogenital tract. In one embodiment, intranasal administration is used for treating pneumonia or otitis media (because it can more effectively suppress pneumococcal nasopharyngeal carriage, thereby attenuating the infection at its initial stage).

[0097] The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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. The amount of conjugate in each vaccine dose can be selected as an amount that elicits an immune protective response without significant adverse effects. Such amounts can vary depending on the pneumococcal serotype. Generally, for polysaccharide-based conjugates, each dose contains 0.1 - 100 μg of each polysaccharide, particularly 0.1 - 10 μg, more particularly 1 - 5 μg of each polysaccharide. For example, each dose can contain 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.

[0098] In one embodiment, the dose of the aluminum salt is 10, 15, 20, 25, 30, 50, 7 0, 100, 125, 150, 200, 300, 500 or 700 μg, or 1, 1.2, 1.5, 2, 3, 5 mg, or more. In yet another embodiment, the dosage of the aluminum salt described above is per 1 μg of the recombinant protein.

[0099] According to any of the methods herein, and in one embodiment, the subject is human. In certain embodiments, the human patient is an infant (less than 1 year old), a toddler (about 12 - 24 months) or a young child (about 2 - 5 years old). In another embodiment, the human patient is an elderly patient (> 65 years old). The compositions of the present invention are further suitable for use in older children, adolescents and adults (e.g., age 18 - 45 years old or 18 - 65 years old).

[0100] In one embodiment of the method of the present invention, the composition of the present invention is administered as a single dose. Another embodiment, the vaccine is administered 2, 3, 4 or more times, with sufficient intervals in between administered. For example, the composition can be administered at intervals of 1, 2, 3, 4, 5 or 6 months, or at intervals of any combination thereof administered. The immunization schedule can follow the schedule designated for pneumococcal vaccines. For example, the daily schedule for infants and toddlers regarding invasive diseases caused by Streptococcus pneumoniae is at 2, 4, 6 and 12 - 15 months of age. Thus, in a preferred embodiment, the composition is administered in a series of 4 doses at 2, 4, 6 and 12 - 15 months of age.

[0101] The compositions herein can further include one or more proteins derived from Streptococcus pneumoniae Examples of Streptococcus pneumoniae proteins suitable for inclusion are those described in the International Patent Those specified in Published Patent Applications WO02 / 083855 and WO02 / 053761, etc. exist.

[0102] Formulation The compositions of the present invention can be administered to a subject by one or more methods known to those skilled in the art (e.g., parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal), and can be formulated accordingly.

[0103] In one embodiment, the composition of the present invention is administered by epidermal injection, intramuscular injection, intravenous injection, intraarterial injection, subcutaneous injection, or intranasal mucosal injection of a liquid preparation. Liquid preparations for injection include solutions, etc.

[0104] The compositions of the present invention can be formulated as single-dose vials, multi-dose vials, or pre-filled syringes.

[0105] In another embodiment, the composition of the present invention is administered orally and is thus formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation. Solid oral preparations include tablets, capsules, pills, granules, pellets, etc. Liquid oral preparations include solutions, suspensions, dispersions, emulsions, oils, etc.

[0106] Pharmaceutically acceptable carriers for liquid preparations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions (e.g., physiological saline and buffered There are, for example, oils (such as those of animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, other fish oils, or lipids derived from milk or eggs). The pharmaceutical composition can be isotonic, hypotonic or hypertonic. However, in many cases, it is preferably essentially isotonic when administered for injection or infusion. Therefore, for storage, the pharmaceutical composition can preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it can be diluted to be isotonic with an isotonic solution before administration.

[0107] The pharmaceutical composition can be isotonic, hypotonic or hypertonic. However, in many cases, it is preferably essentially isotonic when administered for injection or infusion. Therefore, for storage, the pharmaceutical composition can preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it can be diluted to be isotonic with an isotonic solution before administration. The pharmaceutical composition can be isotonic, hypotonic or hypertonic. However, in many cases, it is preferably essentially isotonic when administered for injection or infusion. Therefore, for storage, the pharmaceutical composition can preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it can be diluted to be isotonic with an isotonic solution before administration. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol.

[0108] Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. 2 Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. 2 Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol. Isotonic agents can be ionic isotonic agents such as salts or non-ionic isotonic agents such as carbohydrates. Examples of ionic isotonic agents include, but are not limited to, sodium chloride (NaCl), calcium chloride (CaCl₂), potassium chloride (KCl), and magnesium chloride (MgCl₂). Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol, and glycerol.

[0109] Furthermore, it is preferable that at least one pharmaceutically acceptable additive is a buffering agent. Depending on the purpose, for example, when the pharmaceutical composition is for injection or infusion, in many cases, it is desirable that the composition contains a buffering agent (where the buffering agent can buffer the solution to a pH within the range of 4 to 10 (for example, 5 to 9, for example, 6 to 8)). Furthermore, it is preferable that at least one pharmaceutically acceptable additive is a buffering agent. Depending on the purpose, for example, when the pharmaceutical composition is for injection or infusion, in many cases, it is desirable that the composition contains a buffering agent (where the buffering agent can buffer the solution to a pH within the range of 4 to 10 (for example, 5 to 9, for example, 6 to 8)). Furthermore, it is preferable that at least one pharmaceutically acceptable additive is a buffering agent. Depending on the purpose, for example, when the pharmaceutical composition is for injection or infusion, in many cases, it is desirable that the composition contains a buffering agent (where the buffering agent can buffer the solution to a pH within the range of 4 to 10 (for example, 5 to 9, for example, 6 to 8)). Furthermore, it is preferable that at least one pharmaceutically acceptable additive is a buffering agent. Depending on the purpose, for example, when the pharmaceutical composition is for injection or infusion, in many cases, it is desirable that the composition contains a buffering agent (where the buffering agent can buffer the solution to a pH within the range of 4 to 10 (for example, 5 to 9, for example, 6 to 8)).

[0110] The buffer can be selected from the group consisting of, for example, TRIS buffer, acetate buffer, glutamate buffer, lactate buffer, maleate buffer, tartrate buffer, phosphate buffer, citrate buffer, carbonate buffer, glycinate buffer, histidine buffer, glycine buffer, succinate buffer and triethanolamine buffer.

[0111] The buffer can further be selected, for example, particularly when the pharmaceutical preparation is for parenteral use , from USP-compliant buffers for parenteral use. For example, the buffer can be selected from the group consisting of the following: monobasic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid; dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid; polybasic acids such as , citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine and TRIS.

[0112] Examples of parenteral vehicles (for subcutaneous injection, intravenous injection, intra-arterial injection or intramuscular injection) include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s solution and fixed oils. Examples of intravenous vehicles include fluid replacement solutions and nutritional replacement solutions, electrolyte replacement solutions (e.g., those based on Ringer’s dextrose). Examples thereof are sterile liquids, with or without surfactants and other pharmaceutically acceptable adjuvants added, such as water and oils. Generally, water, physiological saline, aqueous dextrose and related sugar solutions , glycol (e.g., propylene glycol or polyethylene glycol, etc.) is preferred as a liquid carrier, particularly preferred as a liquid carrier for injection solutions. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other fish oils, or lipids derived from milk or eggs.

[0113] The formulations of the present invention may further contain a surfactant. Preferred surfactants include, but are not limited to, the following: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tween); copolymers of ethylene oxide (EO) , propylene oxide (PO) and / or butylene oxide (BO) (sold under the trade name of DO WFAX TM ), for example, linear EO / PO block copolymers ; octoxynol (which can have various numbers of repeating ethoxy (oxy-1,2-ethanediyl) groups, where octoxynol-9 (Triton X-1 00, or t-octylphenoxypolyethoxyethanol) is particularly important); ( octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-4 0); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxy ylates, such as Tergitol NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl TM alcohol, stearyl alcohol and oleyl alcohol (known as Brij surfactants), for example, triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as Span); and, sorbitan esters (generally which is known as SPAN, for example, sorbitan trioleate (Span 85) and sorbitan monolaurate.

[0114] The preferred amount (wt%) of the surfactant is as follows: polyoxyethylene sor bitan esters (e.g., PS80) 0.01 to 1%, in particular, about 0.1%; octyl - or nonylphenoxypolyoxyethanol (e.g., Triton X-100, or other surfactants of the Triton series) 0.001 to 0.1%, in particular, 0.0 05 to 0.02%; polyoxyethylene ethers (e.g., laureth 9) 0.1 to 2 0%, preferably 0.1 to 10%, in particular, 0.1 to 1% or about 0.5%.

[0115] The formulation further contains a pH-buffered physiological saline aqueous solution. The buffer is, for example, TR IS buffer, acetate buffer, glutamate buffer, lactate buffer, maleate buffer tartrate buffer, phosphate buffer, citrate buffer, carbonate buffer, glycinate buffer histidine buffer, glycine buffer, succinate buffer, HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, MOPS (3-(N- morpholino)propanesulfonic acid) buffer, MES (2-(N-morpholino)ethanesulf onic acid) buffer and triethanolamine buffer. The buffer has the ability to buffer the solution to a pH within the range of 4 to 10, 5.2 to 7.5 or 5.8 to 7.0. In a particular embodiment of the invention, the buffer is selected from the group consisting of phosphate, succinate salt, histidine, MES, MOPS, HEPES, acetate or citrate. salt, histidine, MES, MOPS, HEPES, acetate or citrate. is selected. The buffer may further be, for example, in particular, if the pharmaceutical preparation is for parenteral use it can also be selected from USP-compliant buffers for parenteral use. The concentration of the buffer solution is in the range of 1 mM to 50 mM or 5 mM to 50 mM. In certain embodiments, the buffer is 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 embodiments, the histidine is at a final concentration of 20 mM ± 2 mM.

[0116] The aqueous physiological saline solution (i.e., a solution containing NaCl) is preferred, but other salts suitable for the formulation include, but are not limited to, CaCl 2 , KCl and MgCl 2 and combinations thereof. Non-ionic isotonic agents (which include, but are not limited to, sucrose, trehalose, mannitol, sorbitol and glycerol) can be used instead of salts. Suitable ranges of salts include, but are not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one embodiment, the physiological saline is NaCl, which may optionally be present at a concentration of 20 mM to 170 mM. In a preferred embodiment, the formulation contains an L-histidine buffer together with sodium chloride.

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

[0118] In another embodiment, the pharmaceutical composition is delivered by a controlled release system. For example, the drug can be administered using intravenous injection, transdermal patches, liposomes or other administration methods. In another embodiment, a polymeric material is used, for example, in microspheres or implants.

[0119] The composition of the present invention may also include one or more proteins derived from Streptococcus pneumoniae . Examples of Streptococcus pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO02 / 083855 and WO02 / 053761, etc. .

[0120] Analytical Methods Molecular Weight and Concentration of Conjugates Using HPSEC / UV / MALS / RI Assay Analysis Inject the conjugate sample and separate it by high performance size exclusion chromatography (HPSEC ). Detection is performed by a series of ultraviolet (UV) detectors, multi-angle light scattering (MALS ) detectors and refractive index (RI) detectors. Protein concentration is calculated from UV280 using the extinction coefficient. Polysaccharide concentration is analyzed from the RI signal (contribution from both protein and polysaccharide) using the dn / dc coefficient, which is the change in the refractive index of the solution due to the change in solute concentration reported in mL / g. The average molecular weight of the sample is calculated using the measured concentration and the light scattering data for the entire sample peak by Astra software (Wyatt Technology Corporation, Santa Barbara, CA ). For polydisperse molecules, there are many forms of molecular weight averages. For example, number average molecular weight Mn, weight average molecular weight Mw, and z-average molecular weight Mz (Molecules ), 2015, 20:10313-10341). Unless otherwise specified, the term "molecular weight" as used throughout this specification is the weight average molecular weight. ). ). ). ). ). , 2015, 20:10313-10341). Unless otherwise specified, the term "molecular weight" as used throughout this specification is the weight average molecular weight.

[0121] Conjugated Tan as a Measure of the Number of Covalent Bonds between Polysaccharide and Carrier Protein Measurement of Lysine Consumption in Protein Using Waters AccQ-Tag amino acid analysis (AAA), the degree of conjugation in the conjugate sample is measured. The sample is hydrolyzed using gas-phase acid hydrolysis on an Eldex workstation to break down the carrier protein into its component amino acids. The free amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized sample is then analyzed using UPLC with UV detection on a C18 column. The average protein concentration is obtained using representative amino acids other than lysine. The lysine consumption (i.e., lysine loss) during conjugation is determined by the difference between the average measured amount of lysine in the conjugate and the estimated amount of lysine in the starting protein. The free polysaccharides (i.e., polysaccharides not conjugated to CRM197) in the conjugate sample are measured by first precipitating the free protein and conjugate using deoxycholate (DOC) and hydrochloric acid. The precipitate is then filtered off, and the filtrate is analyzed for free polysaccharide concentration by HPSEC / UV / MALS / RI. The free polysaccharides are calculated as the percentage of the total polysaccharides measured by HPSEC / UV / MALS / RI. The free polysaccharides, polysaccharide-CRM197 conjugate, and free CRM197 in the conjugate sample are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. The free amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized sample is then analyzed using UPLC with UV detection on a C18 column. The average protein concentration is obtained using representative amino acids other than lysine. The lysine consumption (i.e., lysine loss) during conjugation is determined by the difference between the average measured amount of lysine in the conjugate and the estimated amount of lysine in the starting protein. The free polysaccharides (i.e., polysaccharides not conjugated to CRM197) in the conjugate sample are measured by first precipitating the free protein and conjugate using deoxycholate (DOC) and hydrochloric acid. The precipitate is then filtered off, and the filtrate is analyzed for free polysaccharide concentration by HPSEC / UV / MALS / RI.

[0122] Free Polysaccharide Test The free polysaccharides (i.e., polysaccharides not conjugated to CRM197) in the conjugate sample are measured by first precipitating the free protein and conjugate using deoxycholate (DOC) and hydrochloric acid. The precipitate is then filtered off, and the filtrate is analyzed for free polysaccharide concentration by HPSEC / UV / MALS / RI. The free polysaccharides are calculated as the percentage of the total polysaccharides measured by HPSEC / UV / MALS / RI. The free polysaccharides, polysaccharide-CRM197 conjugate, and free CRM197 in the conjugate sample are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. The free polysaccharides are calculated as the percentage of the total polysaccharides measured by HPSEC / UV / MALS / RI. The free polysaccharides are calculated as the percentage of the total polysaccharides measured by HPSEC / UV / MALS / RI.

[0123] Free Protein Test The free polysaccharides, polysaccharide-CRM197 conjugate, and free CRM197 in the conjugate sample are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. The free polysaccharides, polysaccharide-CRM197 conjugate, and free CRM197 in the conjugate sample are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. - Separate by electrophoresis. Briefly, mix the sample with an MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3, and separate it using a pre-conditioned bare-fused silica capillary (bare-fused silica cap illary). Monitor the separation at 200 nm and quantify the free CRM197 using a CRM197 standard curve. Report the results of the free protein as a percentage (%) of the total protein content determined by the HPSEC / UV / MALS / RI procedure.

[0124] Polysaccharide Activation Degree Assay Conjugation occurs by reductive amination mainly between the activated aldehyde and the lysine residues of the carrier protein. The level of activation as the molar number of aldehydes per mole of polysaccharide repeating unit is important for controlling the conjugation reaction.

[0125] In this assay, derivatize the polysaccharide with 2.5 mg / mL thiosemicarbazide ( TSC) at pH 4.0 to introduce a chromophore (use 1.25 mg / mL TSC for the derivatization of activated polysaccharides for serotypes 1, 5, 9V). Allow the derivatization reaction to proceed to reach a plateau. The actual time varies depending on the reaction rate of each serotype. Then, separate the TSC-Ps from TSC and other low molecular weight components by high performance size exclusion chromatography. Detect the signal by UV absorbance at 266 nm. Calculate the level of activated aldehyde relative to the injection of a Mono-TSC standard curve or by directly using a predetermined attenuation factor. Mono-TSC is a synthesized thiosemicarb arbazide of monosaccharide It is a bazone derivative. Then, the aldehyde level is determined using the Ps concentration measured by HPSEC / UV / MALS / RI assay and converted to the number of moles of aldehyde per mole of repeating unit (Ald / RU).

[0126] Although various embodiments of the present invention have been described with reference to the accompanying specification, the present invention is not limited to the embodiments described in detail, and those skilled in the art can make various changes and modifications without departing from the scope or spirit of the present invention as defined in the appended claims.

Examples

[0127] The present invention is illustrated by the following examples, but the present invention is not limited by these examples.

[0128] Example 1 Preparation of Streptococcus pneumoniae 35B Capsular Polysaccharide Fermentation Methods for culturing pneumococci are well known in the art. See, for example, "Chase, 1967, Methods of Immunology and Immunochemistry 1:52". Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP0497524B1. The processes described below generally follow the methods described in European Patent No. EP0497524B1 and are generally applicable to all pneumococcal serotypes, except where specifically modified. se, 1967, Methods of Immunology and Immu nochemistry 1:52". See also European Patent No. EP0497524B1 for methods of preparing pneumococcal capsular polysaccharides. The processes described below generally follow the methods described in European Patent No. EP0497524B1 and are generally applicable to all pneumococcal serotypes, except where specifically modified. 1. 7524B 1 and generally apply to all pneumococcal serotypes, except where specifically modified.

[0129] A strain of Streptococcus pneumoniae serotype 35B was obtained from the “Merck Culture Collecti on”. If necessary, subtypes can be identified based on the Quelling reaction using specific antisera. See, for example, U.S. Patent No. 5,847,112. The resulting isolates were further cloned by serial plating on agar plates consisting of an animal component-free medium containing no hemin and containing soy peptone, yeast extract, and glucose in two steps. The clonal isolates for each serotype were further propagated in liquid culture using an animal component-free medium containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare a pre-master cell bank. For example, refer to U.S. Patent No. 5,847,112. The obtained isolates were further cloned by continuously plating on agar plates consisting of an animal component-free medium that does not contain hemin and contains soy peptone, yeast extract, and glucose in two steps. The clonal isolates for each serotype were further propagated in liquid culture using an animal component-free medium containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare a pre-master cell bank. The production of Streptococcus pneumoniae serotype 35B consisted of cell growth and batch manufacturing fermentation followed by chemical inactivation and subsequent downstream purification. The thawed cell bank vials were grown using a shaking flask or culture bottle containing a sterile animal component-free growth medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, and glucose. The cell growth culture was grown in a sealed shaking flask or bottle with minimized gas exchange under temperature and agitation control. After achieving a specific culture density measured by the optical density at 600 nm, a portion of the cell growth culture was transferred to a medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, sodium chloride, potassium phosphate, and glucose. The obtained isolates were further cloned by serial plating on agar plates consisting of an animal component-free medium containing no hemin and containing soy peptone, yeast extract, and glucose in two steps. The clonal isolates for each serotype were further propagated in liquid culture using an animal component-free medium containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare a pre-master cell bank. The production of Streptococcus pneumoniae serotype 35B consisted of cell growth and batch manufacturing fermentation followed by chemical inactivation and subsequent downstream purification. The thawed cell bank vials were grown using a shaking flask or culture bottle containing a sterile animal component-free growth medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, and glucose. The cell growth culture was grown in a sealed shaking flask or bottle with minimized gas exchange under temperature and agitation control.

[0130] After achieving a specific culture density measured by the optical density at 600 nm, a portion of the cell growth culture was transferred to a medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, sodium chloride, potassium phosphate, and glucose. The production of Streptococcus pneumoniae serotype 35B consisted of cell growth and batch manufacturing fermentation followed by chemical inactivation and subsequent downstream purification. The thawed cell bank vials were grown using a shaking flask or culture bottle containing a sterile animal component-free growth medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, and glucose. The cell growth culture was grown in a sealed shaking flask or bottle with minimized gas exchange under temperature and agitation control. After achieving a specific culture density measured by the optical density at 600 nm, a portion of the cell growth culture was transferred to a medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, sodium chloride, potassium phosphate, and glucose. The cell growth culture was grown in a sealed shaking flask or bottle with minimized gas exchange under temperature and agitation control. After achieving a specific culture density measured by the optical density at 600 nm, a portion of the cell growth culture was transferred to a medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, sodium chloride, potassium phosphate, and glucose. After achieving a specific culture density measured by the optical density at 600 nm, a portion of the cell growth culture was transferred to a medium containing soy peptone or ultrafiltered soy peptone, yeast extract or ultrafiltered yeast extract, sodium chloride, potassium phosphate, and glucose. The cell growth culture was grown in a sealed shaking flask or bottle with minimized gas exchange under temperature and agitation control. It was transferred to a production fermenter containing a sterilized animal component-free growth medium. Temperature, pH, pressure, and agitation were controlled. Since no sparging was used, the air flow overlie was also controlled.

[0131] When the glucose was almost depleted, the fermentation was terminated by adding phenol, a chemical inactivator, to the batch. Pure phenol was added to a final concentration of 0.8 - 1.2% to inactivate the cells and release the capsular polysaccharide from the cell wall. The primary inactivation was carried out for a specified time in the fermenter while controlling the continuation of temperature and agitation. After primary inactivation, the batch was transferred to another container where it was held under controlled temperature and agitation for an additional specified time to complete the inactivation. Completion of inactivation was confirmed by either microbial plating techniques or verification of phenol concentration and specified time. Next, the inactivated broth was purified.

[0132] Purification of Ps The purification of pneumococcal polysaccharide consisted of several centrifugation, depth filtration, concentration / diafiltration operations, and precipitation steps. All procedures were carried out at room temperature unless otherwise indicated.

[0133] The inactivated broth from the fermenter culture of Streptococcus pneumoniae was aggregated with cationic polymers (e.g., BPA - 1000, Petrolite “Tretolite” and “Spectru m 8160” and poly(ethyleneimine), “Millipore pDADMAC” combined. After the aggregation stage and the aging period, the aggregated solids were removed by centrifugation and multiple deep filtration steps The broth free of impurities was concentrated and diafiltered using a 100 kDa - 500 kDa MW CO (molecular weight cut-off) filter. The dia filtration was carried out using Tris, MgCl 2 buffer and sodium phosphate buffer The residual nucleic acids and proteins were removed by diafiltration

[0134] Furthermore, impurity removal was performed by reprecipitating the polysaccharide in sodium acetate and phenol using denatured alcohol and / or iso propanol. During the phenol precipitation step, sodium acetate and phenol ( liquefied phenols or solid phenols) in sodium phosphate saline buffer were added to the diafiltered retention fluid Next, alcohol fractionation of the polysaccharide was carried out in two steps. In the first step, low percentage alcohol was added to the preparation to precipitate cell debris and other unwanted impurities, but the crude polysaccharide remained in solution. The impurities were removed by a deep filtration step. Then, additional isopropanol or denatured alcohol was added to the batch to recover the polysaccharide from the solution The precipitated polysaccharide pellet was recovered by centrifugation, ground, dried as a powder and stored frozen at -70 °C

[0135] Example 2 Activation of Streptococcus pneumoniae Serotype 35B Polysaccharide The purified pneumococcal capsular Ps powder was dissolved in water and filtered through 0.45 microns. The dissolved polysaccharide was homogenized to reduce the viscosity of the Ps solution. The homogenization pressure and the homogenizer​ The number of passes through was controlled to 100 bar / 5 passes. The homogenized polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0136] The polysaccharide solution was adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by adding a 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.01, 0.03, 0.05, 0.07, 0.09 or 0.11 moles of sodium metaperiodate per mole of polysaccharide repeating unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). The oxidation reaction proceeded for 1 hour at 22 °C. The activated polysaccharide was dialyzed against 10 mM potassium phosphate, pH 6.4 at approximately 4 °C and subsequently dialyzed against distilled water for a total of 3 days using a 5 kDa NMWCO dialysis cassette.

[0137] As shown in Table 1, when sodium metaperiodate is added to the activation reaction, the size of the Streptococcus pneumoniae serotype 35B polysaccharide chain decreases while the number of aldehydes per repeating unit increases. As the molar equivalent of sodium metaperiodate (charged to the reaction) increases, the size of the 35B polysaccharide decreases and the number of reactive aldehydes per repeating unit increases. This suggests that activation cleaves the main chain of the polysaccharide at the acyclic triol sites.

Table 2

[0138] Example 3 Conjugation of Streptococcus pneumoniae Serotype 35B Polysaccharide to CRM197 Activation of Polysaccharide The polysaccharides were activated and purified as described in Example 2.

[0139] Conjugation of Polysaccharide to CRM197 Purified CRM197 (which was obtained by expression in Pseudomonas fluorescens as previously described (WO2012 / 17387 6A1)) was diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 micron. The activated polysaccharides were formulated for lyophilization at 6 mg Ps / mL using a 5% w / v sucrose concentration. CRM197 was formulated for lyophilization at 6 mg Pr / mL using a 1% w / v sucrose concentration.

[0140] The formulated solutions of Ps and CRM197 were lyophilized separately. The lyophilized Ps and CRM197 materials were separately redissolved in an equal volume of DMSO. In the case of the arm containing salt, sodium chloride was spiked into the dissolved Ps to a concentration of 10 mM sodium chloride. The solutions of polysaccharide and CRM197 were mixed to give a polysaccharide concentration of 6 g Ps / L (arms 1

[0141] ~6) or 7.5 g Ps / L (arms 7 - 12) and a mass ratio of polysaccharide to CRM197 of 3. The conjugation was continued at 34 °C for 3 hours. Following the conjugation reaction, sodium borohydride (per mole of polysaccharide repeating unit ~6) or 7.5 g Ps / L (arms 7 - 12) and a mass ratio of polysaccharide to CRM197 of 3. The conjugation was continued at 34 °C for 3 hours. 3. The conjugation was continued at 34 °C for 3 hours.

[0142] Reduction with Sodium Borohydride Following the conjugation reaction, sodium borohydride (per mole of polysaccharide repeating unit 2 moles) was added and incubated at 34 °C for 1 hour. The batch was diluted with 150 mM sodium chloride containing approximately 0 .025% (w / v) polysorbate 20 at approximately 4 °C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 150 mM sodium chloride, 0.05% (w / v) polysorbate 20 at approximately 4 °C for 3 days using a 300 kDa NMWCO dialysis cassette.

[0143] As shown in Table 2, the number of reactive aldehydes per repeating unit and the size of the Streptococcus pneumoniae serotype 35B polysaccharide chain (which are controlled by the amount of periodate loaded during the activation step ) directly affect the properties of the 35B conjugate. As the size of the oxidized 35 B polysaccharide increases, the number of reactive aldehydes per repeating unit decreases. Therefore, oxidized 35B polysaccharide molecules with fewer aldehydes per repeating unit result in larger-sized 3 5B conjugates, but (1) reduce lysine consumption, (2) increase the proportion of free polysaccharide (%), and (3) increase the proportion of free protein (%). On the other hand, oxidized 35B polysaccharide molecules with an increased number of aldehydes per repeating unit have a smaller polysaccharide size and result in 35B conjugates with a size less than 1000 kD.

[0144] It should be noted that dialysis is not very effective in removing free protein and free polysaccharide compared to full-scale purification methods such as ultrafiltration (see Table 8 for examples of conjugates purified using ultrafiltration). The values of free polysaccharide and free protein in Table 2 are used to show the trend of conjugation efficiency in relation to the activation level of the polysaccharide .

Table 3

[0145] Example 4 Effect of Temperature on the Attributes of Streptococcus pneumoniae Serotype 35B Polysaccharide-Protein Conjugate Effect Activation of Polysaccharide The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through a 0.45 micron filter. The dissolved polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0146] The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by the addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeating unit in order to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). The oxidation reaction proceeded for 1 hour at 22 °C.

[0147] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, and subsequently diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The ultrafiltration was carried out at 2 - 8 °C.

[0148] Conjugation of Polysaccharide to CRM197 Purified CRM197 (which, as previously described (WO2012 / 17387 6A1), Pseudomonas fluorescens obtained by expressing in (scens)) was tangentialized with a 5 kDa NMWCO Diafiltered against 2 mM phosphate, pH 7.2 buffer using a sharp flow ultrafiltration membrane and filtered through 0.2 microns.

[0149] The activated polysaccharide was formulated for lyophilization at 6 mg Ps / mL using a sucrose concentration of 5% w / v and a sodium chloride concentration of 10 mM. CRM197 was formulated for lyophilization at 6 mg Pr / mL using a sucrose concentration of 1% w / v.

[0150] The formulated solutions of Ps and CRM197 were individually lyophilized. The lyophilized Ps and CRM197 materials were individually redissolved in an equal volume of DMSO. The solutions of the polysaccharide and CRM197 were mixed to a polysaccharide concentration of 6 g Ps / L and a mass ratio of polysaccharide to CRM197 of 3. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeating unit) was added and the conjugation reaction proceeded at 28 °C, 30 °C, 34 °C or 38 °C for 3 hours.

[0151] Reduction with Sodium Borohydride Following the conjugation reaction, sodium borohydride (2 mol per mol of polysaccharide repeating unit) was added and incubated for 1 hour at the same temperature as the conjugation. The batch was diluted at about 4 °C with 150 mM sodium chloride containing about 0.025% (w / v) polysorbate 20. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed using a 300 kDa MWCO dialysis cassette against 10 mM histidine in 150 mM sodium chloride, pH 7.0 containing 0.015% (w / v) polysorbate 20. ​​​​​​​​​​​​​It was dialyzed at 2 - 8°C for 3 days.

[0152] As shown in Table 3, the temperature used during the conjugation reaction affects the properties of the Streptococcus pneumoniae 35B polysaccharide / CRM197 conjugate. When the conjugation temperature rises from 22°C to 38°C, the free polysaccharide fraction in the 35B conjugate decreases. This suggests that conjugation is more effective at higher temperatures within this range.

Table 4

[0153] Example 5 Effect of Moisture Concentration on the Attributes of Streptococcus pneumoniae Serotype 35B Polysaccharide-Protein Conjugate Effect The conjugation of Streptococcus pneumoniae serotype 35B polysaccharide to CRM197 is carried out in an organic solvent such as DMSO. Even in an organic environment, if other components are added, water may be introduced into the conjugation reaction (for example, spiking in a reducing agent, or, since DMSO is hygroscopic, over time). The effect of water content on the properties of the Streptococcus pneumoniae serotype 35B polysaccharide - protein (CRM197) conjugate was investigated by spiking distilled water at the start of conjugation.

[0154] Activation of Polysaccharide The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through a 0.45 - micron filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0155] ​​​​​​​​​​​Next, the polysaccharide solution was adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by adding 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 mol of sodium metaperiodate per mole of polysaccharide repeating unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). The oxidation reaction proceeded at 22 °C for 2 hours. The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, and subsequently diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was carried out at 2 - 8 °C.

[0156] Purified CRM197 (which was obtained by expression in Pseudomonas fluorescens as previously described (WO2012 / 17387 6A1)) was diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 micron. The activated polysaccharide was formulated for lyophilization at 6 mg Ps / mL using a 5% w / v sucrose concentration. CRM197 was formulated for lyophilization at 6 mg Pr / mL using a 1% w / v sucrose concentration.

[0157] Conjugation of Polysaccharide to CRM197 The formulated solutions of Ps and CRM197 were lyophilized separately. The lyophilized Ps

[0158]

[0159] And the CRM197 material was individually redissolved in an equal volume of DMSO. The polysaccharide and CRM 197 solutions were mixed to a polysaccharide concentration of 6 g Ps / L and a polysaccharide to CRM197 mass ratio of 3. Water was spiked immediately until the target percentage was reached in the conjugation reaction. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. The conjugation reaction proceeded at 34 °C for 3 hours.

[0160] Reduction with Sodium Borohydride Following the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added and incubated at 34 °C for 1 hour. The batch was diluted with 150 mM sodium chloride containing approximately 0 .025% (w / v) polysorbate 20 at approximately 4 °C. Potassium phosphate buffer was then added to neutralize the pH. The batch was dialyzed against 10 mM histidine in 150 mM sodium chloride, pH 7.0, containing 0.015% (w / v) polysorbate 20 at 2 - 8 °C for 3 days using a 300 kDa MWCO dialysis cassette. As shown in Table 4, free polysaccharides and free proteins increase with the increasing water content in the conjugation reaction. This is probably due to protein aggregation in high concentrations of water, and thus care must be taken to minimize the water content during the conjugation reaction for effective conjugation.

[0161] As shown in Table 4, free polysaccharides and free proteins increase with the increasing water content in the conjugation reaction. This is probably due to protein aggregation in high concentrations of water, and thus care must be taken to minimize the water content during the conjugation reaction for effective conjugation.

Table 5

[0162] Example 6 ​​​​​​ Preparation of Streptococcus pneumoniae Serotype 35B Polysaccharide-Protein Conjugate Containing Sodium Chloride in Lyophilized Formulation Preparation To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide. To improve the attributes of the conjugate, salts can be spiked into the conjugation reaction, but this also introduces water into the conjugation reaction. As shown in Example 5 above, water promotes the aggregation of proteins and polysaccharides, thereby interfering with the serotype 35B polysaccharide conjugation reaction. The water content during the conjugation reaction was minimized by including sodium chloride in the formulation prior to lyophilization. Reduction of the water content during the conjugation reaction resulted in conjugates with increased size, increased loss of lysine, and reduced free protein and free polysaccharide.

[0163] Activation of Polysaccharide The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through a 0.45 micron filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through a 0.45 micron filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through a 0.45 micron filter. The filtered dissolved polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0164] The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C. The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C. The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C. The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C. The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C. The polysaccharide solution was then adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by addition of a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded for 2 hours at 22 °C.

[0165] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 and then diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane Ultrafiltration was performed at 2 - 8 °C.

[0166] Conjugation of Polysaccharide to CRM197 Purified CRM197 (which was obtained by expression in Pseudomonas fluorescens as previously described (WO2012 / 17387 6A1)) was diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 micron. The activated polysaccharide was formulated for lyophilization at 6 mg Ps / mL using a 5% w / v sucrose concentration. Various levels of sodium chloride were spiked into Ps-PreLyo for the specific arms described in Table 5. CRM197 was formulated for lyophilization at 6 mg Pr / mL using a 1% w /

[0167] v sucrose concentration. The formulated solutions of Ps and CRM197 were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved separately in an equal volume of DMSO. The solutions of the polysaccharide and CRM 197 were mixed to give a polysaccharide concentration of 6 g Ps / L, a mass ratio of polysaccharide to CRM197 of 3, and a sodium chloride concentration of 0 mM, 5 mM, 10 mM or 15 mM

[0168] as described in Table 5, the addition of sodium chloride was Ps pre-lyo as described in Table 5, the addition of sodium chloride was Ps pre-lyo and CRM197 of 3, and a sodium chloride concentration of 0 mM, 5 mM, 10 mM or 15 mM and a sodium chloride concentration of 0 mM, 5 mM, 10 mM or 15 mM As described in Table 5, the addition of sodium chloride was Ps pre-lyo either via or by spiking the Ps-DMSO with an aqueous solution after Ps dissolution This was carried out. The conjugation reaction proceeded at 34 °C for 3 hours.

[0169] Reduction with Sodium Borohydride Following the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added and incubated at 34 °C for 1 hour. The batch was diluted with 150 mM sodium chloride containing approximately 0 .025% (w / v) polysorbate 20 at approximately 4 °C. Next, potassium phosphate buffer was added to neutralize the pH. The batch was dialyzed against 10 mM histidine in 150 mM sodium chloride, pH 7.0, containing 0.015% (w / v) polysorbate 20 at 2 - 8 °C for 3 days using a 300 kDa MWCO dialysis cassette. As shown in Table 5, spiking the conjugation reaction with aqueous sodium chloride (arms 2 - 4) gives conjugate attributes similar to those of the conjugate without sodium chloride (arm 1 ). Conversely, adding sodium chloride to the reaction before lyophilization (arms 5 - 7) produces a conjugate with an increased Mw and reduced free Ps compared to the state without sodium chloride.

[0170] As described below, pneumococcal serotype 35B polysaccharide was dissolved and chemically

Table 6

[0171] Example 7 Preparation of Streptococcus pneumoniae Serotype 35B Polysaccharide-Protein Conjugate for Mouse Testing Preparation As described below, pneumococcal serotype 35B polysaccharide was dissolved and chemically ​​​​​activated and the buffer was exchanged by ultrafiltration. The activated polysaccharide and purified CRM197 were each lyophilized and redissolved in DMSO. Then, the solutions of the redissolved polysaccharide and CRM197 were combined and conjugated. The resulting conjugate was purified by ultrafiltration and finally filtered through 0.2 microns. To generate a conjugate with desired attributes, several process parameters (e.g., pH, temperature, concentration, and time) within each step were controlled.

[0172] Activation of Polysaccharide The purified pneumococcal serotype 35B capsular Ps powder was dissolved in water and filtered through 0.45 microns. When appropriate, the dissolved polysaccharide was homogenized to reduce the viscosity of the Ps solution. The homogenization pressure and the number of passes through the homogenizer were controlled to reduce the viscosity of the polysaccharide. The polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

[0173] Then, the polysaccharide solution was adjusted to 22 °C and pH 5 using sodium acetate buffer. Activation of the polysaccharide was initiated by adding a 10 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was 0.038 or 0.047 moles of sodium metaperiodate per mole of polysaccharide repeat unit to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeat unit). The oxidation reaction proceeded at 22 °C for 1 - 2 hours.

[0174] The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4. Rationed and subsequently diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane. Ultrafiltration was performed at 2 - 8 °C.

[0175] Conjugation of Polysaccharide to CRM197 Purified CRM197 (which was obtained by expression in Pseudomonas fluorescens as previously described (WO2012 / 17387 6A1)) was diafiltered against 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through 0.2 micron. The activated polysaccharide was formulated for lyophilization at 6 mg Ps / mL using a 5% w / v sucrose concentration and for group 6 using sodium chloride. CRM197 was formulated for lyophilization at 6 mg Pr / mL using a 1% w / v sucrose concentration.

[0176] The formulated Ps and CRM197 solutions were individually lyophilized. The lyophilized Ps and CRM197 materials were individually redissolved in an equal volume of DMSO. In groups 3, 4 and 8, sodium chloride was spiked into the Ps - DMSO solution. The solutions of the polysaccharide and CRM197 were mixed to a polysaccharide concentration of 6 g Ps / L and a polysaccharide to CRM197 loading

[0177] mass ratio of 1.5, 2.2 or 3.0. The conjugation reaction was allowed to proceed at 34 °C for 3 - 6 hours. The conjugation parameters are summarized in Table 6. 8, sodium chloride was spiked into the Ps - DMSO solution. The solutions of the polysaccharide and CRM19 7 were mixed to a polysaccharide concentration of 6 g Ps / L and a polysaccharide to CRM197 loading mass ratio of 1.5, 2.2 or 3.0. The conjugation reaction was allowed to proceed at 34 °C for 3 - 6 hours. The conjugation parameters are summarized in Table 6.

[0178] Reduction with Sodium Borohydride Following the conjugation reaction, sodium borohydride (per mole of polysaccharide repeating unit) was added. The batch was then cooled to about 4° C. and incubated for 1 hour at 34° C. for about 0.2 hours. Diluted in 150 mM sodium chloride containing .025% (w / v) polysorbate 20. The pH was then neutralized by adding potassium phosphate buffer. The batch was concentrated and cooled to 300k. Da NMWCO tangential flow ultrafiltration membrane was used to filter 0.015% (w / v ) 10 mM Histidine in 150 mM sodium chloride containing polysorbate 20, pH 7.0 The mixture was diafiltered against cysteine ​​at 2-8°C.

[0179] Final Filtration and Product Storage The retentate batch was filtered through 0.5 / 0.2 micron and then diluted with 0.015% (w / v) poly An additional 10 mM hyaluronate in 150 mM sodium chloride containing resorbate 20, pH 7.0 The resulting mixture was diluted with sucrose, dispensed into aliquots, and frozen at or below -60°C. The properties of the conjugates obtained are summarized in Table 7. [Table 7] [Table 8]

[0180] Example 8 Formulation of Pneumococcal Conjugate Vaccine for Mouse Testing Individual 35B-CRMs prepared using the various processes described in the Examples above The 197 conjugate was used in the formulation of a monovalent pneumococcal conjugate vaccine.

[0181] The monovalent drug is prepared using the pneumococcal polysaccharide 35B-CRM197 conjugate. The target concentration of pneumococcal polysaccharide antigen was 4.0 μg / mL, and the concentration was measured using 20 mM histidine, pH 5.8, and and 150 mM sodium chloride and 0.1% w / v polysorbate-20 (PS-20). For mouse studies groups 7 and 8, the formulations were formulated in Prepare using 250 μg [Al] / mL in the form of aluminum phosphate.

[0182] Required volume of bulk conjugate required to achieve target concentrations of individual pneumococcal polysaccharide antigens was calculated based on the batch volume and the concentration of the individual bulk polysaccharides. Add 100 mg of histidine to a solution of sodium chloride and PS-20 to obtain 2x the amount of conjugate. The blend was generated. The formulation vessel containing the 2x conjugate blend was placed under magnetic stirring. Mix using a bar and then sterile filter into a separate container. The adjugate blend was then mixed into a separate container containing an aluminum phosphate adjuvant (APA). The desired polysaccharides, excipients and APs can be added to the vessel or diluted with saline. Achieve a concentration of A (if necessary). The formulation is then filled into glass vials or syringes. Fill and store at 2-8°C.

[0183] Example 9 Effect of Conjugate / Formulation Process on the Immunogenicity of 35B-CRM197 Vaccine Effect Young female CD1 mice (6-8 weeks old, n=5 / group) were cultured on days 0, 14 and 2 On day 8, the mice were immunized with 0.1 mL of the 35B-CRM197 vaccine prepared as above. The 35B-CRM197 vaccine was administered without APA adjuvant (Granules 1 and 2) for each immunization. (Loops 1-6) or 25 μg of APA (Groups 7 and 8) were added to CRM197. The subjects were administered 0.4 μg of 35B polysaccharide conjugated with 35B polysaccharide (Tables 7 and 8). Sera were collected on day 35 (pre-infection) and day 35 (post-dose 3, PD3) by trained animal care staff. Mice were observed at least daily by the staff for any signs of disease or distress. The vaccine formulation in vivo was found to be safe, with no vaccine-related adverse events observed. All animal studies were performed in accordance with the Guide for Care and Use of Laboratory Animals of the Na in strict accordance with the National Institutes of Health recommendations. The mouse experiment protocol was conducted using the " Institutional Animal Care and Use Commit Approved by "tee".

[0184] Mouse sera were assayed for anti-PnPs35B IgG titers using ELISA as previously described. A(Chen ZF et al, BMC Infectious Diseases e, 2018, 18: 613), and anti-35B functional antibodies were evaluated. For details, please refer to the previously described protocol (www.vaccine.uab.edu). Based on the opsonophagocytosis assay (OPA) and owned by University of University of Alabama (UAB) Research Foundation The evaluation was performed using Opsotiter® 3 software licensed for use with the Burton, RL, Nahm MH, Clin Vaccine Immuno l 2006, 13:1004-9; Burton, RL, Nahm MH, Clin Vaccine Immunol 2012, 19:835-41). Immunity Pre-immune sera were assayed as pools and PD3 sera were assayed individually.

[0185] There was no detectable anti-35B IgG at a 1:200 dilution in pre-immune sera (data not shown). However, IgG titers increased with PD3 for all vaccine formulations (Figure 1A). The data also further showed that different conjugation / formulation processes had a significant impact on the immunogenicity of the 35B polysaccharide-CRM197 vaccine . Adding the APA adjuvant increased the IgG titer compared to the vaccine without adjuvant (Group 7 vs. Group 1 / Group 8 vs. Group 4). Adding 5 mM NaCl to the conjugation reaction also increased the IgG titer of the 35B-CRM197 vaccine (Group 6 vs. Group 5). The anti-35B OPA titer followed the same trend as seen in the IgG titer (Figure 1B).

[0186] Serotype 35B polysaccharide-CRM197 conjugates with attributes ranging as shown in Table 7 were found to be immunogenic.

Claims

1. Streptococcus pneumoniae serotype 35B with a molecular weight of 1,000 kDa to 7,000 kDa Sugar-protein conjugates.

2. The conjugate is a 3 mol / mol protein to 9 mol / mol protein The conjugate of claim 1 , comprising lysine consumption.

3. The conjugate is 4 mol / mol protein to 8 mol / mol protein The conjugate of claim 1 , comprising lysine consumption.

4. A composition comprising the conjugate of claim 2 or 3, further comprising: Free polysaccharides less than 30% of total polysaccharides and free protein less than 30% of total protein The composition comprising:

5. A composition comprising the conjugate of claim 2 or 3, further comprising: Free polysaccharides less than 20% of total polysaccharides and free protein less than 20% of total protein The composition comprising:

6. The conjugate according to claims 1 to 3, wherein the protein is CRM197.

7. The protein component of the polysaccharide-protein conjugate is CRM197. Item 6. The composition according to item 4 or 5.

8. A process for preparing the conjugates of claims 1 to 3, comprising: wherein the activation comprises activating the polysaccharide repeat unit at a concentration of 0.01 mole per mole of polysaccharide repeat unit. The process using periodate in the range of 0.1 moles of periodate.

9. The range of periodate is from 0.03 to 0.06 moles of periodate per mole of polysaccharide repeat unit.

9. The process of claim 8, wherein the iodate is a salt of iodate.

10. 10. The process of claim 8 or 9, wherein the periodate is sodium periodate.

11. The process according to claim 8 or 9, wherein the periodate is sodium metaperiodate. Seth.

12. A process for making the conjugates of claims 1 to 3, comprising: Conjugating to a protein, wherein the conjugation comprises The process is carried out at a conjugation temperature of 22°C to 38°C.

13. 13. The process of claim 12, wherein the conjugation temperature is from 32°C to 36°C.

14. A process for preparing the conjugates of claims 1 to 3, comprising: wherein the activation is at 0.01 to 0.1 moles per mole of polysaccharide repeating unit. and mixing the polysaccharide with the protein. conjugating to a conjugate of and performing the process at a jugation temperature.

15. A process for preparing the conjugates of claims 1 to 3, comprising: wherein said activation is at a concentration of 0.03 to 0.06 moles per mole of polysaccharide repeating unit. (using a periodate in the range of 100 to 200% periodate) and conjugating the antibody to a substrate (wherein the conjugation is performed at 32° C. to 36° C.) (c) at a bonding temperature.

16. 16. The method according to claim 14 or 15, wherein the conjugation is carried out in an aprotic solvent. The process of installation.

17. 17. The process of claim 16, wherein the aprotic solvent is DMSO.

18. 18. The method according to claim 16 or 17, wherein the conjugation is carried out in the presence of sodium chloride. The process described.

19. 19. The process of claim 18, wherein the concentration of sodium chloride is 5 to 15 mM.

20. Any of claims 16 to 19, wherein the solvent contains less than 1.2% (v / v) water. The process described in

21. 21. The process of claim 20, wherein the solvent contains less than 0.6% (v / v) water. 。

22. 22. The process of claim 21, wherein the solvent contains less than 0.3% (v / v) water. 。

23. The conjugation is performed at an aldehyde per repeat unit in the range of 0.01 to 0.

1. The process according to any one of claims 12 to 22, which is carried out using an activated polysaccharide comprising Process.

24. The aldehyde per repeating unit is in the range of 0.03 to 0.

06. The process of installation.

25. The conjugation is carried out using an activated polysaccharide having a molecular weight in the range of 30-200 KDa. The process according to any one of claims 12 to 24, which is carried out using a compound of the formula:

26. The process according to claim 25, wherein the molecular weight range of the activated polysaccharide is 40 to 100 KDa. Seth.

27. An immunogenic multivalent pneumococcal conjugate vaccine composition comprising any one of claims 8 to 26. The serotype 35B pneumococcus polysaccharide-protein complex prepared by the process described herein is The immunogenic multivalent pneumococcal conjugate vaccine composition comprising a serotype 1 or 2 conjugate.

28. An immunogenic multivalent pneumococcal conjugate vaccine composition comprising the serotypes of claims 1 to 3. The immunogenic polysaccharide-protein conjugate comprises 35B Streptococcus pneumoniae polysaccharide-protein conjugate.

5. A pneumococcal conjugate vaccine composition.

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