Preparation method for conjugating pneumococcal polysaccharide to carrier protein

Abstract

The present invention provides a number of process improvements related to the conjugation of capsular polysaccharides from Streptococcus pneumoniae to carrier proteins. These methods are serotype specific and include acid hydrolysis, the addition of sodium chloride to the reductive amination reaction, and the addition of sucrose to solubilize the polysaccharide. The polysaccharide-protein conjugates prepared using the methods of the present invention can be included in a multivalent pneumococcal conjugate vaccine.

Description

Technical Field

[0001] The present invention provides a number of process improvements related to the conjugation of capsular polysaccharides from S. pneumoniae to carrier proteins. The polysaccharide-protein conjugates prepared using the methods of the present invention may be included in a multivalent pneumococcal conjugate vaccine. Background Art

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

[0003] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, has proven to be invaluable in preventing pneumococcal disease in adults, especially the elderly and high-risk groups. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides are T-cell-independent immunogens that induce weak or no responses in infants. Chemical conjugation of bacterial polysaccharide immunogens to carrier proteins can convert the infant's immune response to a T-cell-dependent one. Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM 197 Described as carrier proteins for bacterial polysaccharide immunogens due to the presence of T-cell stimulating epitopes in their amino acid sequences.

[0004] Pneumococcal conjugate vaccine First licensed in the United States in February 2000, it contains the seven most common serotypes of pneumococcal disease that cause invasive pneumococcal disease in infants and young children at the time (4, 6B, 9V, 14, 18C, 19F, and 23F). Afterwards, due to In some parts of the world, however, invasive pneumococcal disease in children has been significantly reduced due to the presence of serotypes in the The serotype coverage is limited, and there is evidence of some emerging serotypes in the United States (e.g., 19A, etc.). See 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., 2007, J Infect Dis 196:1346-54; Traore et al., 2009, Clin Infect Dis 48:S181-S189.

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

[0006] Current multivalent pneumococcal conjugate vaccines have been effective in reducing the incidence of pneumococcal disease associated with those serotypes present in the vaccine. However, the prevalence of pneumococci expressing serotypes not present in the vaccine is increasing. Process conditions for novel serotypes must be determined for each serotype with respect to conjugation efficiency and for certain serotypes that present unique challenges. Therefore, improved process conditions are needed for conjugating new pneumococcal serotypes for inclusion in future vaccines. Summary of the invention

[0007] The present invention provides a number of process variations in the preparation of polysaccharides (Ps) from Streptococcus pneumoniae that are unique to specific serotypes. These process variations improve the properties of the polysaccharide and / or the solubility of the polysaccharide, resulting in better conjugation.

[0008] In one embodiment, the present invention provides process conditions for obtaining size-reduced pneumococcal polysaccharides from serotypes 12F, 23A, 24F and 31, which, when conjugated to a carrier protein (Pr) in an aprotic solvent, exhibit desirable conjugate properties. Specifically, reducing the size of Ps of these serotypes by acid hydrolysis compared to homogenization can produce lower Ps molecular weights for protein conjugation, thereby improving conjugate properties such as lysine consumption, free Ps or free Pr.

[0009] In one embodiment, the present invention provides process conditions for obtaining improved polysaccharide-protein conjugate properties after conjugation using sodium chloride in an aprotic solvent (e.g., DMSO), particularly for S. pneumoniae polysaccharides from serotypes 15A, 16F, 17F, 20, 24F, and 35B. Specifically, in this embodiment, the inclusion of ≥1 mM sodium chloride before or during the conjugation reaction (regardless of where in the process the sodium chloride is added) improves conjugate properties, such as larger conjugate size, higher lysine consumption, lower free PS or free Pr.

[0010] In one embodiment, the present invention provides a series of pre-lyophilization preparation conditions for pneumococcal polysaccharides of serotypes 3, 8 and 24F to ensure complete dissolution after lyophilization. Specifically, the polysaccharide is formulated with sucrose and water so that the mass ratio of sucrose to polysaccharide is ≥30 times, and optimally ≥40 times. For example, for a given pre-lyophilization polysaccharide concentration of 2 mg Ps / mL, the sucrose concentration should be a minimum of 60 mg sucrose / mL (6% w / v sucrose), and optimally ≥80 mg sucrose / mL (8% w / v sucrose) for dissolution after lyophilization.

[0011] Polysaccharides formulated in these ways can be conjugated to proteins following lyophilization and reconstitution to produce the desired properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The effect of sodium chloride on the conjugate size of a polysaccharide from S. pneumoniae serotype 20 is shown.

[0013] Figure 2 The effect of sodium chloride on lysine consumption of a polysaccharide from S. pneumoniae serotype 20 is shown.

[0014] Figure 3 The effect of sodium chloride on free Ps and Pr of S. pneumoniae polysaccharide from serotype 20 is shown. DETAILED DESCRIPTION

[0015] The present invention provides a number of process variations in the preparation of polysaccharides (Ps) from Streptococcus pneumoniae that are unique to specific serotypes. These process variations improve the properties of the polysaccharide and / or the solubility of the polysaccharide, resulting in better conjugation.

[0016] The inventors have discovered that, as shown in the Examples, size reduction of certain S. pneumoniae polysaccharides by acid hydrolysis prior to protein conjugation results in improved conjugate properties. Without being bound by any particular theory, one possible mechanism for the use of acid hydrolysis that may explain the observed behavior is that conjugation with the lower molecular weight Ps produced by acid hydrolysis provides less steric hindrance between the Ps and Pr molecules during the conjugation reaction, which may in turn lead to enhanced interactions and improved conjugation.

[0017] The inventors have discovered that complete dissolution of certain S. pneumoniae polysaccharides requires the presence of sucrose after lyophilization, as shown in the Examples. Without being bound by any particular theory, one possible mechanism for the use of sucrose that may explain the observed dissolution behavior is that certain polysaccharide serotypes, due to their chemical structure, are more prone to self-associate during lyophilization or dissolution than other serotypes, and that a higher sucrose / polysaccharide ratio inhibits self-association, allowing dissolution after lyophilization.

[0018] The inventors have found that the addition of ≥1 mM sodium chloride, either before or during the conjugation reaction, particularly for pneumococcal polysaccharides from serotypes 16F, 20 and 24F, results in improved conjugate properties, such as larger conjugate size, higher lysine consumption, lower free Ps or free Pr, regardless of where in the process the sodium chloride is added, as shown in the examples. Without being bound by any particular theory, one possible mechanism for the use of sodium chloride may explain the observed conjugation behavior for sodium chloride, namely that for certain polysaccharide serotypes, sodium chloride provides electrostatic shielding due to its chemical structure and charge distribution, thereby allowing enhanced interaction with the protein and resulting in improved conjugation. Sodium chloride also provides additional ionic strength to the reaction mixture, which can reduce exposure of hydrophobic regions of the carrier protein, thereby reducing protein aggregation.

[0019] As used herein, the term "polysaccharide" (Ps) refers to any antigenic carbohydrate element (or antigenic unit) commonly used in the field of immunization and bacterial vaccines, including but not limited to "sugar", "oligosaccharide", "polysaccharide", "liposaccharide", "lipo-oligosaccharide (LOS)", "lipopolysaccharide (LPS)", "glycosylate", "glycoconjugate", "derivatized or activated polysaccharide or oligosaccharide", etc. Unless otherwise specified, the polysaccharide nomenclature used herein follows the IUB-IUPAC Joint Commission on Biochemical Nomenclature (JCBM) Recommendations 1980. See JCBN, 1982, J. Biol. Chem. 257: 3352-3354.

[0020] As used herein, "immunogenic composition" refers to a composition containing an antigen, such as a bacterial capsular polysaccharide or a polysaccharide-protein conjugate, which has the ability to induce an immune response in a host, such as a mammal, mediated by body fluids or cells or both. The immunogenic composition can be used to sensitize the host by presenting an antigen associated with an MHC molecule on the cell surface. In addition, antigen-specific T cells or antibodies can be produced to protect the immunized host in the future. Therefore, the immunogenic composition can protect the host from bacterial infection, reduce severity, or protect the host from death caused by bacterial infection. The immunogenic composition can also be used to produce polyclonal or monoclonal antibodies, which can be used to confer passive immunity to a subject. The immunogenic composition can also be used to produce functional antibodies, such as measured by bacterial killing in an animal efficacy model or by an opsonized phagocyte killing assay.

[0021] As used herein, the term "isolated" in relation to polysaccharides refers to the separation of pneumococcal serotype-specific capsular polysaccharides from purified polysaccharides using purification techniques known in the art, including the use of centrifugation, depth filtration, precipitation, ultrafiltration, activated carbon treatment, diafiltration and / or column chromatography. Typically, isolated polysaccharides refer to partial removal of proteins, nucleic acids and non-specific endogenous polysaccharides (C-polysaccharides). Isolated polysaccharides contain less than 10%, 8%, 6%, 4% or 2% protein impurities and / or nucleic acids. Relative to type-specific polysaccharides, isolated polysaccharides contain less than 20% C-polysaccharides.

[0022] As used herein, the term "purified" in relation to bacterial capsular polysaccharides refers to purification of the polysaccharide from cell lysate by methods such as centrifugation, precipitation and ultrafiltration. Typically, purified polysaccharides are those that have been freed from cell debris and DNA.

[0023] As used herein, the term "Mw" refers to weight average molecular weight and is typically expressed in Da or kDa. Mw takes into account that larger molecules contain more of the total mass of a polymer sample than smaller molecules. Mw can be determined by techniques such as static light scattering, small angle neutron scattering, X-ray scattering, and sedimentation velocity.

[0024] As used herein, the term "Mn" refers to the number average molecular weight and is usually 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 determined by methods such as gel permeation chromatography, viscometry by (Mark-Houwink equation), colligative methods such as vapor pressure osmometry, end group determination or proton NMR. Mw / Mn reflects polydispersity.

[0025] As used herein, the term "comprising" when used with the immunogenic compositions of the invention refers to the inclusion of any other ingredients (for antigenic mixtures, subject to the limitations of the "consisting of" language), such as adjuvants and excipients. When used with a multivalent polysaccharide-protein conjugate mixture, the term "consisting of" refers to a mixture with those specific pneumococcal polysaccharide-protein conjugates and without other pneumococcal polysaccharide-protein conjugates from different serotypes.

[0026] Unless otherwise stated, all ranges provided herein are inclusive of the recited lower and upper limits.

[0027] Capsular polysaccharide

[0028] Capsular polysaccharides of Streptococcus pneumoniae from serotypes of the present invention (e.g., serotypes 23A, 24F, and 31) can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria, and polysaccharides can be sized to a certain extent by known methods (see, e.g., European Patent Nos. EP497524 and EP497525); and preferably by microfluidization using a homogenizer or chemical hydrolysis. In one embodiment, Streptococcus pneumoniae strains corresponding to each polysaccharide serotype are grown in a soybean-based culture medium. Various polysaccharides are then purified by standard steps (including centrifugation, precipitation, and ultrafiltration). See, e.g., U.S. Patent Application Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,112. Polysaccharides can be sized to reduce viscosity and / or improve the filterability of subsequent conjugated products. Chemical hydrolysis can be performed using acetic acid. Mechanical sizing can be performed using high-pressure homogenization shearing.

[0029] For serotypes 12F, 23A, 24F and 31, it was found that homogenization of polysaccharides from these serotypes did not produce the desired properties. See Example 3. Acid hydrolysis can be performed by heating the polysaccharide batch to 80-92°C (preferably 90°C for serotypes other than 12F), adding an acid (e.g., acetic acid, hydrochloric acid, phosphoric acid, citric acid) to a final concentration of 50-200 mM, and then incubating for at least 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes. In certain embodiments, the acid hydrolysis is performed for up to 90 minutes, up to 150 minutes, or up to 155 minutes. At the end of the incubation period, the batch is neutralized by adding, for example, concentrated potassium phosphate pH 7 buffer to a final concentration of 400 mM and cooling to ≤22°C. The size-reduced polysaccharide is filtered at 0.2 microns, then concentrated and diafiltered with water using a 5-10 kDa NMWC tangential flow ultrafiltration membrane.

[0030] In some embodiments, the molecular weight of the purified polysaccharide prior to conjugation is between 5 kDa and 4,000 kDa. The molecular weight can be calculated by size exclusion chromatography (SEC) in conjunction with a multi-angle light scattering detector (MALS) and a refractive index detector (RI). In other such embodiments, the average molecular weight of the polysaccharide is between 10 kDa and 4,000 kDa; between 50 kDa and 4,000 kDa; between 50 kDa and 3,000 kDa; between 50 kDa and 2,000 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 4,000 kDa; between 100 kDa and 3,000 kDa; between 100 kDa and kDa; 2,000 kDa; 100 kDa to 1,500 kDa; 100 kDa to 1,000 kDa; 100 kDa to 750 kDa; 100 kDa to 500 kDa; 100 to 400 kDa; 200 kDa to 4,000 kDa; 200 kDa to 3,000 kDa; 200 kDa to 2,000 kDa; 200 kDa to 1,500 kDa; 200 kDa to 1,000 kDa; or 200 kDa and 500 kDa. In certain embodiments, the average molecular weight is 50-300 kDa.

[0031] In certain embodiments, the polysaccharide has 10 to 10,000, 10 to 5,000, 10 to 4,000, or 10 to 1000 repeating units. In certain aspects, the polysaccharide has 20 to 400, 30 to 300, 40 to 200, or 50 to 100 repeating units. In certain aspects, the polysaccharide has 40 to 900 repeating units.

[0032] Carrier Protein

[0033] Polysaccharides from one or more of the S. pneumoniae serotypes described herein may be conjugated to a carrier protein to improve immunogenicity in children, the elderly, and / or immunocompromised subjects. Where more than one serotype is used in a multivalent composition, the serotypes may be prepared with the same carrier protein or different carrier proteins. Typically each capsular polysaccharide of the same serotype is conjugated to the same carrier protein.

[0034] In a specific embodiment of the invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic variant of diphtheria toxin (DT). The CRM197 carrier protein is a mutant form of DT, which is made non-toxic by a single amino acid substitution at residue 52 in fragment A. In one embodiment, the CRM197 carrier protein is isolated from a culture of Corynebacterium diphtheriae strain C7 (β197) grown in casamino acids and yeast extract-based medium. In another embodiment, CRM197 is recombinantly prepared according to the method described in U.S. Patent No. 5,614,382. Typically, CRM197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, Pfenex Expression Technology TM CRM197 in Pseudomonas fluorescens was prepared (Pfenex Inc., San Diego, CA).

[0035] Other suitable carrier proteins include additional inactivated bacterial toxins, such as DT, diphtheria toxoid fragment B (DTFB), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), E. coli LT (heat-labile enterotoxin), E. coli ST (heat-stable enterotoxin), and exotoxin A of Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; see International Patent Application Publication No. WO 02 / 091998), pneumococcal adhesin protein (PsaA), C5a peptidase from group A or group B streptococci, or Haemophilus influenzae protein D, pneumolysin (Kuo et al., 1995, Infect Immun 63:2706-13), including ply detoxified in some way, such as dPLY-GMBS (see International Patent Application Publication No. WO 04 / 081515) or dPLY-formol, PhtX, including fusions of PhtA, PhtB, PhtD, PhtE and Pht proteins, such as PhtDE fusions, PhtBE fusions (see International Patent Application Publication Nos. WO 01 / 98334 and WO 03 / 54007). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (from Neisseria meningitidis), PD (Haemophilus influenzae protein D; see, e.g., European Patent No. EP 0 594 610B) or immunologically functional equivalents thereof, synthetic peptides (see European Patent Nos. EP 0 378 881 and EP 0 427 347), heat shock proteins (see International Patent Application Publication Nos. WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see International Patent Application Publication No. WO 98 / 58668 and European Patent No. EP 0 471 177), cytokines, lymphokines, growth factors or hormones (see International Patent Application Publication No. WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from antigens derived from various pathogens (see Falugi et al., 2001, Eur J Immunol 31:3816-3824), such as N19 protein (see Baraldoi et al., 2004, Infect Immun 72:4884-7), iron uptake protein (see International Patent Application Publication No. WO 01 / 72337), toxin A or B of Clostridium difficile (C. difficile) (see International Patent Publication No. WO 00 / 61761) and flagellin (see Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.

[0036] Other DT mutants can also be used as carrier proteins, such as CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844), CRM9, CRM45, CRM102, CRM103 and CRM107, and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; Glu-148 deletion or mutation to Asp, Gln or Ser and / or Ala 158 mutation to Gly and other mutations described in U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations described in U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragments described in U.S. Pat. No. 5,843,711.

[0037] In the case of using a multivalent vaccine, the second carrier protein can be used for one or more antigens. The second carrier protein is preferably a protein that is non-toxic and non-reactive and can be obtained in sufficient amount and purity. The second carrier protein is also conjugated or connected to an antigen such as Streptococcus pneumoniae polysaccharide to enhance the immunogenicity of the antigen. The carrier protein should comply with standard conjugation procedures. In one embodiment, each capsular polysaccharide that is not conjugated to the first carrier protein is conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule is conjugated to a single carrier protein). In another embodiment, the capsular polysaccharide that is not conjugated to the first carrier protein is conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to a single carrier protein). In such an embodiment, each capsular polysaccharide of the same serotype is usually conjugated to the same carrier protein.

[0038] Conjugation

[0039] Pneumococcal polysaccharides are typically conjugated to proteins by reductive amination in an aprotic solvent such as DMSO. The activated polysaccharide (Ps) and protein (Pr) are typically lyophilized, resuspended in DMSO, and then mixed with added sodium cyanoborohydride and sodium borohydride to achieve conjugation. The process details are provided below.

[0040] For many pneumococcal serotypes, this process produced conjugates that met the target attributes of size, lysine consumption, free polysaccharide, and free protein. However, for some serotypes, it was found that even by optimizing conjugation parameters such as Ps and Pr concentrations and conjugation time, it was more difficult to achieve the target conjugate attributes using the DMSO method. See the Examples. As described below, the present invention provides several solutions to overcome these problems.

[0041] Prior to conjugation, the purified polysaccharide can be chemically activated to enable the sugar to react with the carrier protein to form an activated polysaccharide. As used herein, the term "activated polysaccharide" refers to a polysaccharide that has been chemically modified as described below to enable conjugation to a linker or a carrier protein. The purified polysaccharide can be optionally connected to a linker. Once activated or connected to a linker, each capsular polysaccharide is conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugate can be prepared by known coupling techniques.

[0042] In certain embodiments, a polysaccharide can be coupled to a joint to form a polysaccharide-joint intermediate, wherein the free end of the joint is an ester group. Thus, a joint is a joint in which at least one end is an ester group. The other end is selected so that it can react with the polysaccharide to form a polysaccharide-joint intermediate.

[0043] In certain embodiments, the polysaccharide can be coupled to the joint using the primary amine groups in the polysaccharide. In this case, the joint usually has an ester group at both ends. This allows one of the ester groups to react with the primary amine groups in the polysaccharide to be coupled by nucleophilic acyl substitution. The reaction produces a polysaccharide-joint intermediate, in which the polysaccharide is connected to the joint by an amide bond. Therefore, the joint is a bifunctional joint, which provides a first ester group for reacting with the primary amine groups in the polysaccharide and a second ester group for reacting with the primary amine groups in the carrier molecule. A typical joint is adipic acid N-hydroxysuccinimide diester (SIDEA).

[0044] In certain embodiments, coupling can also occur indirectly, i.e., by another linker for derivatizing the polysaccharide before coupling to the linker. The polysaccharide is coupled to another linker using a carbonyl group at the reducing end of the polysaccharide. The coupling comprises two steps: (a1) reacting the carbonyl group with another linker; and (a2) reacting the free end of another linker with the linker. In these embodiments, another linker generally has a primary amine group at both ends, thereby allowing step (a1) to react one of the primary amine groups with the carbonyl group in the polysaccharide by reductive amination. A primary amine group that reacts with the carbonyl group in the polysaccharide is used. Hydrazide or hydroxylamino are suitable. The same primary amine group is generally present at both ends of another linker. The reaction produces a polysaccharide-another linker intermediate, in which the polysaccharide is coupled to another linker by a CN bond.

[0045] In certain embodiments, different groups in polysaccharides, particularly carboxyl groups, can be used to couple polysaccharides to other joints. The coupling includes two steps: (a1) reacting a group with another joint; and (a2) reacting the free end of another joint with a joint. In this case, the other joints generally have primary amine groups at both ends, thereby allowing step (a1) to react one of the primary amine groups with the carboxyl groups in the polysaccharide by EDAC activation. Primary amine groups reacting with the carboxyl groups activated by EDAC in the polysaccharide are used. Hydrazide groups are suitable. The same primary amine groups are generally present at both ends of another joint. The reaction produces a polysaccharide-other joint intermediate, in which the polysaccharide is coupled to another joint by an amide bond.

[0046] In one embodiment, chemical activation of the polysaccharide and subsequent conjugation to the carrier protein by reductive amination can be achieved by methods described in U.S. Pat. Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 184072, 2007 / 0231340 and 2007 / 0184071, and International Patent Application Publication Nos. WO2006 / 110381, WO2008 / 079653 and WO2008 / 143709. The chemical reaction may require activation of the pneumococcal polysaccharide in the presence of an oxidizing agent by reaction with any oxidizing agent that converts the primary hydroxyl group to an aldehyde (e.g., TEMPO) (WO2104 / 097099), or by reacting two vicinal hydroxyl groups to an aldehyde, such as a periodate (including sodium periodate, potassium periodate or periodic acid). The reaction results in random oxidation of the primary hydroxyl groups of the carbohydrate or random oxidative cleavage of the vicinal hydroxyl groups of the carbohydrate to form reactive aldehyde groups.

[0047] In this embodiment, the lysyl group of protein is coupled to the carrier protein by direct amination reduction. For example, by reacting a mixture of activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride, optionally conjugated in the presence of nickel for aqueous conjugation. The conjugation reaction can be carried out in the presence of an aqueous solution or dimethyl sulfoxide (DMSO). See, for example, U.S. Patent Application Publication No. US2015 / 0231270 and US2011 / 0195086 and European Patent No. EP 0471177 B1. Then unreacted aldehyde is capped by adding a strong reducing agent (such as sodium borohydride).

[0048] Reductive amination involves two steps, (1) oxidation of the polysaccharide to form a reactive aldehyde, and (2) reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein to form a stable amine conjugate bond. Prior to oxidation, the polysaccharide is optionally size-reduced. Mechanical methods (e.g., homogenization) or chemical hydrolysis may be employed. Chemical hydrolysis may be performed using acetic acid. The oxidation step may involve reaction with a periodate. For the purposes of the present invention, the term "periodate" includes periodate and periodic acid. The term also includes metaperiodate (104 - ) and orthoperiodate (IO6 5- ), and includes various salts of periodate (e.g. sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably sodium periodate (NaIO4). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably periodic acid.

[0049] In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide free radical compound, such as a piperidine-N-oxyl or pyrrolidine-N-oxyl compound, which selectively oxidizes primary hydroxyl groups in the presence of an oxidant (e.g., as described in International Patent Application Publication No. WO 2014 / 097099). In the reaction, in the catalytic cycle, the actual oxidant is an N-oxoammonium salt. In one aspect, the stable nitroxyl or nitroxide free radical compound is a piperidine-N-oxyl or pyrrolidine-N-oxyl compound. In one aspect, the stable nitroxyl or nitroxide free radical compound carries a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl free radical compound is TEMPO or a derivative thereof. In one aspect, the oxidizing agent is a molecule with an N-halogenated moiety. In one aspect, the oxidant is selected from N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

[0050] In certain aspects, the oxidant is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as a co-oxidant (as described in International Patent Application Publication No. WO2014 / 097099). Thus, in one aspect, a saccharide conjugate from Streptococcus pneumoniae can be obtained by a method comprising the following steps: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; b) reacting the activated saccharide with a carrier protein comprising one or more amine groups (the method is hereinafter referred to as "TEMPO / NCS-reductive amination").

[0051] Optionally, the oxidation reaction is quenched by adding a quencher. The quencher can be selected from a vicinal diol, a 1,2-amino alcohol, an amino acid, glutathione, a sulfite, a bisulfate, a dithionite, a metabisulfite, a thiosulfate, a phosphite, a hypophosphite or a phosphorous acid (e.g., glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid).

[0052] The second step of the reductive amination conjugation process is to use a reducing agent to reduce the imine (Schiff base) bond between the activated polysaccharide and the carrier protein to form a stable conjugated bond (so-called reductive amination). Suitable reducing agents include cyanoborohydrides (e.g., sodium cyanoborohydride) or sodium borohydride. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0053] In certain embodiments of the methods of the present invention, the reductive amination reaction is carried out in an aprotic solvent (or a mixture of aprotic solvents). In one embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. If lyophilized, DMSO or DMF solvent can be used to reconstitute the activated polysaccharide and carrier protein. In one embodiment, the aprotic solvent is DMSO.

[0054] At a sucrose:Ps mass ratio of 25 times, up to 5% sucrose has been used to achieve optimal solubility in DMSO after lyophilization. See, for example, International Patent Application Publication No. WO2017 / 013548. For Streptococcus pneumoniae polysaccharides obtained from serotypes 3, 8, and 24F, it was found that higher levels of sucrose were required to fully dissolve the polysaccharides prior to protein conjugation in aprotic solvents. In some embodiments, for these serotypes, a sucrose concentration greater than 5% in aqueous solution is used. In some embodiments, for these serotypes, a sucrose:Ps mass ratio greater than 25 times is used, such as at least 30 times, at least 35 times, or at least 40 times. In some embodiments, the mass ratio of sucrose to polysaccharide before lyophilization is greater than or equal to 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 or more. Other sugars, such as trehalose or mannitol, may be used.

[0055] It has also been found that the presence of sodium chloride (NaCl) during the protein conjugation process, whether in aqueous solution or in an aprotic solvent, can be used to reduce free protein levels, increase conjugate molecular weight, reduce free polysaccharide and / or increase lysine consumption of pneumococcal polysaccharides purified from serotypes 15A, 16F, 17F, 20, 23A, 24F and 35B. Thus, the present invention can protect a method for preparing a polysaccharide-protein conjugate, the method comprising reacting a pneumococcal polysaccharide in a first solution with a protein in a second solution to form a third solution, wherein the polysaccharide-protein conjugate reaction occurs to form a polysaccharide-protein conjugate, wherein the third solution comprises at least 1 mM salt.

[0056] Sodium chloride can be added anywhere in the conjugation process, from preparing the polysaccharide and protein for lyophilization before the conjugation reaction to the conjugation reaction itself, such as during the Schiff base reaction or during the reductive amination process in the presence of sodium cyanoborohydride. In some embodiments, the polysaccharide and protein are lyophilized separately, and the salt can be added to the polysaccharide solution (first solution) or the protein solution (second solution) or both. In some embodiments, the polysaccharide and protein are lyophilized from the same solution to which the salt is added (i.e., the first solution and the second solution are the same). In some embodiments, the salt is added to the solution (i.e., the third solution) in which the polysaccharide-protein conjugation reaction occurs.

[0057] Other salts may be used, such as other sodium salts, potassium salts such as potassium chloride, lithium salts, magnesium salts, and calcium salts. In some embodiments, 1 mM to 100 mM sodium chloride is added to the dissolved solution of the polysaccharide in the presence of sodium cyanoborohydride during the Schiff base reaction or during the reductive amination reaction. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM sodium chloride is used. In some aspects of these embodiments, no more than 100, 75, or 50 mM sodium chloride is used.

[0058] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped or quenched using a suitable capping agent or quenching agent. In one embodiment, the capping agent or quenching agent is sodium borohydride (NaBH4). Suitable alternatives include sodium triacetoxyborohydride or sodium borohydride or zinc borohydride in the presence of a Bronsted acid or a Lewis acid, an amine borane, such as pyridine borane, 2-pyridinoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (PEMB) or a borohydride exchange resin.

[0059] Sugar conjugates prepared using reductive amination in aprotic solvents are commonly used in multivalent pneumococcal protein conjugate vaccines. Thus, in certain embodiments, for multivalent compositions where not all serotypes are prepared in aprotic solvents, the reduction reaction for the remaining serotypes is carried out in an aqueous solvent (e.g., selected from PBS (phosphate buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-tris, ADA (N-(2-acetamido)iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2 -aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), DIPSO (3-bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), N-bicine or HEPB, pH between 6.0 and 8.5, 7.0 and 8.0 or 7.0 and 7.5).

[0060] In some embodiments, the glycoconjugates of the present invention comprise polysaccharides having a molecular weight between 10 kDa and 10,000 kDa. In other such embodiments, the molecular weight of the polysaccharide is between 25 kDa and 5,000 kDa. In other such embodiments, the molecular weight of the polysaccharide is between 50 kDa and 1,000 kDa. In other such embodiments, the molecular weight of the polysaccharide is between 70 kDa and 900 kDa. In other such embodiments, the molecular weight of the polysaccharide is between 100 kDa and 800 kDa. In other such embodiments, the molecular weight of the polysaccharide is between 200 kDa and 600 kDa. In further such embodiments, the molecular weight of the polysaccharide is 100 kDa to 1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1,000 kDa; 150 kDa to 900 kDa; 1 50kDa to 800kDa; 150kDa to 700kDa; 150kDa to 600kDa; 150kDa to 500kDa; 150kDa to 400kDa; 150kDa to 300kDa; 200kDa to 1,000kDa; 200kDa to 900kDa; 200kDa to 800kDa; 200kDa to 700kDa; 200kDa to 600kDa; 200kDa to 500kDa kDa; 200kDa to 400kDa; 200kDa to 300; 250kDa to 1,000kDa; 250kDa to 900kDa; 250kDa to 800kDa; 250kDa to 700kDa; 250kDa to 600kDa; 250kDa to 500kDa; 250kDa to 400kDa; 250kDa to 350kDa; 300kDa to 1,000kDa; 300kDa kDa; 300 kDa to 800 kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1,000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa. In certain embodiments employing acid hydrolysis, the molecular weight of the polysaccharide is 10 kDa to 200 kDa, 25 kDa to 200 kDa, 50 kDa to 200 kDa, 10 kDa to 150 kDa, 25 kDa to 150 kDa, or 50 kDa to 150 kDa.

[0061] Suitable alternative chemical methods include activating sugars with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate. Therefore, activated sugars can be coupled to amino groups on carrier proteins directly or through spacer (linker) groups. For example, the spacer can be cystamine or cysteamine to obtain thiolated polysaccharides, which can be coupled to carriers by thioether bonds obtained after reacting with maleimide-activated carrier proteins (e.g., using GMBS) or haloacetylated carrier proteins (e.g., using iodoacetimides (e.g., hydrochloric acid iodoacetamide) or N-succinimidyl bromoacetate or SIAB, or SIA or SBAP)). Preferably, cyanate (optionally prepared by CDAP chemistry) is coupled to hexamethylenediamine or adipic acid dihydrazide (ADH), and amino-derived sugars are conjugated to carrier proteins via carboxyl groups on protein carriers using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described in International Patent Application Publication Nos. WO 93 / 15760, WO 95 / 08348, and WO 96 / 29094; and in Chu et al., 1983, Infect. Immunity 40:245-256.

[0062] Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in International Patent Application Publication No. WO 98 / 42721. Conjugation may involve a carbonyl linker, which may be formed by reacting the free hydroxyl groups of the sugar with CDI (see Bethell et al., 1979, J. Biol. Chem. 254: 2572-4; Hearn et al., 1981, J. Chromatogr. 218: 509-18), followed by reaction with the protein to form a carbamate bond. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optionally protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate and coupling the CDI carbamate intermediate to an amino group on the protein.

[0063] After conjugation (reduction reaction and optional capping or quenching reaction), the glycoconjugate can be purified (enriched relative to the amount of polysaccharide-protein conjugate) by a variety of techniques known to the skilled person. These techniques include dialysis, concentration / diafiltration procedures, tangential flow filtration, ultrafiltration, precipitation / elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and depth filtration. See, for example, U.S. Patent No. 6,146,902. In one embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

[0064] One method of characterizing the glycoconjugates of the invention is by the number of lysine residues in the carrier protein (e.g., CRM197) that become conjugated to the sugar, which can be characterized as a series of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent bonding to the polysaccharide can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate material. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15, or between 10 and 12. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the invention is between 7 and 12. In some such embodiments, the carrier protein is CRM197.

[0065] The glycoconjugates of the present invention can also be characterized by the ratio of sugar to carrier protein (weight / weight). In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w / w) is 0.5 to 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the ratio of sugar to carrier protein (w / w) is 0.5 to 2.0, 0.5 to 1.5, 0.8 to 1.2, 0.5 to 1.0, 1.0 to 1.5 or 1.0 to 2.0. In other embodiments, the ratio of sugar to carrier protein (w / w) is 0.8 to 1.2. In a preferred embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is 1-2. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the present invention may contain free sugars that are not covalently conjugated to the carrier protein but are still present in the glycoconjugate composition. The free sugars may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound, adsorbed or captured therein or captured with it).

[0066] In a preferred embodiment, the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 20% free polysaccharide compared to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 15% free polysaccharide compared to the total amount of polysaccharide.

[0067] Multivalent polysaccharide-protein conjugate vaccine

[0068] The polysaccharide-protein conjugate prepared using the method of the present invention can be used for a multivalent polysaccharide-protein conjugate vaccine. In certain embodiments, the multivalent polysaccharide-protein conjugate vaccine comprises one or more of pneumococcal capsular polysaccharides from pneumococcal serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 as free polysaccharides, components of polysaccharide-protein conjugates or combinations thereof to provide a multivalent pneumococcal vaccine. In certain embodiments, the immunogenic composition comprises, consists essentially of, or consists of a capsular polysaccharide from Streptococcus pneumoniae serotype 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44, each conjugated to one or more carrier proteins. Preferably, the saccharide from a particular serotype is not conjugated to more than one carrier protein.

[0069] After purification of the various saccharide conjugates, they are complexed to formulate the immunogenic compositions of the invention. These pneumococcal conjugates are prepared by separate processes and formulated in bulk as single dose formulations.

[0070] Drug / vaccine combinations

[0071] The present invention also provides compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising, essentially consisting of, or consisting of any of the polysaccharide pneumococcal serotype combinations described above and a pharmaceutically acceptable carrier and adjuvant.

[0072] The preparation of polysaccharide-protein conjugates can be accomplished using methods recognized in the art. For example, the respective pneumococcal conjugates can be formulated with a physiologically acceptable carrier to prepare a composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and glucose solutions.

[0073] In a preferred formulation, the vaccine composition is formulated in an L-histidine buffer containing sodium chloride.

[0074] As defined herein, an "adjuvant" is a substance used to enhance the immunogenicity of the immunogenic composition of the present invention. When administered alone, an immunoadjuvant can enhance the immune response to an antigen that is poorly immunogenic, for example, not induce antibody titers or cell-mediated immune responses or induce weak antibody titers or cell-mediated immune responses, increase the titer of antibodies to the antigen and / or reduce the dose of antigen that is effective in achieving an individual immune response. Therefore, adjuvants are commonly used to enhance immune responses 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:

[0075] (1) Aluminum salts (aluminum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

[0076] (2) Oil-in-water emulsion formulations (with or without other specific immunostimulants, such as murine amido peptides (defined below) or bacterial cell wall components), such as (a) MF59 (International Patent Application Publication No. WO 90 / 14837), which contains 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing various amounts of MTP-PE) and is formulated into submicron particles using a microfluidizer, such as a Model 110Y Microfluidizer (Microfluidics, Newton, MA), (b) SAF, which contains 10% squalene, 0.4% Tween 80, 5% pluronic block polymer L121 and thr-MDP, which are microfluidized into submicron emulsions or vortexed to produce larger particle size emulsions, (c) Ribi TM Adjuvant system (RAS), (Corixa, Hamilton, MT), comprising 2% squalene, 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of: 3-O-deacylated monophospholipid A (MPL) described in U.S. Pat. No. 4,912,094; TM ), trehalose dicarboxylate (TDM) and cell wall skeleton (CWS), preferably MPL + CWS (Detox TM ); and (d) Montanide ISA;

[0077] (3) Saponin adjuvants such as Quil A or STIMULON can be usedTM QS-21 (Antigenics, Framingham, MA) (see, e.g., U.S. Pat. No. 5,057,540), or particles derived therefrom, such as ISCOM (immunostimulatory complexes formed by a combination of cholesterol, saponin, phospholipids, and amphiphilic proteins) and (having essentially the same structure as ISCOM, but without the protein);

[0078] (4) bacterial lipopolysaccharide, synthetic lipid A analogs, such as aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa and described in U.S. Pat. No. 6,113,918; one AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, also known as 529 (formerly RC529), which is formulated in aqueous form or a stable emulsion;

[0079] (5) synthetic polynucleotides, such as oligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646);

[0080] (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; and

[0081] (7) Complement, such as a trimer of complement component C3d.

[0082] In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, such as SBAS2 (an oil-in-water emulsion that also contains 3-deacylated monophosphoryl lipid A and QS21).

[0083] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-n-muramyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), and the like.

[0084] In certain embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant can be an alum precipitated vaccine or an alum adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143: 489-493). Aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), hydrated aluminum, aluminum trihydrate, Superfos, Aluminum(III) hydroxide, aluminum hydroxyphosphate (aluminum phosphate adjuvant (APA)), amorphous alumina, alumina trihydrate or aluminum trihydroxy.

[0085] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is manufactured by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After the blending process, the size of the material is reduced with a high shear mixer to obtain a monodisperse particle size distribution. The product is then diafiltered with saline and steam sterilized.

[0086] In certain embodiments, commercially available Al(OH)3 (e.g., or Superhydros or Superfos (Denmark / AccurateChemical and Scientific Co., Westbury, NY) adsorb proteins. In another embodiment, the adsorption of proteins depends on the pI (isoelectric pH) of the protein and the pH of the culture medium. Proteins with lower pIs adsorb positively charged aluminum ions more strongly than proteins with higher pIs. Aluminum salts can establish an Ag reserve that is slowly released over 2-3 weeks, participating in nonspecific activation of macrophages and complement activation, and / or stimulating innate immune mechanisms (possibly by stimulating uric acid). See, e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.

[0087] Typically, the monovalent bulk aqueous conjugate is mixed together and diluted. After dilution, the batch is sterile filtered. Aluminum phosphate adjuvant is aseptically added to give a final concentration of 4 μg / mL for all S. pneumoniae serotypes except serotype 6B (which is diluted to a target of 8 μg / mL) and a final aluminum concentration of 250 μg / mL. The adjuvanted batch is filled into vials or syringes.

[0088] 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 can be obtained from Coley Pharmaceutical Group.

[0089] "CpG-containing nucleotides", "CpG-containing oligonucleotides", "CpG oligonucleotides" and similar terms refer to nucleotide molecules of 6-50 nucleotides in length and containing unmethylated CpG moieties. See, for example, Wang et al., 2003, Vaccine 21: 4297. In another embodiment, any other art-accepted definition of the term is included. CpG-containing oligonucleotides include modified oligonucleotides using any synthetic internucleoside linkages, modified bases and / or modified sugars.

[0090] Methods using CpG oligonucleotides are known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110.

[0091] Administration / dosage

[0092] By administering the vaccine via a systemic or mucosal route, the compositions and formulations described herein can be used to protect or treat people who are susceptible to infection (e.g., pneumococcal infection). For example, the compositions and formulations described herein can be used in a method of inducing an immune response to a pneumococcal capsular polysaccharide conjugate, the method comprising administering to a person an immunogenic composition or formulation described herein in an immunologically effective amount. In another example, the compositions and formulations described herein can be used in a method of vaccinating a person to resist pneumococcal infection, the method comprising the steps of administering to a person an immunogenic composition or formulation described herein in an immunologically effective amount.

[0093] The optimal amounts of the components of a particular vaccine can be determined by standard studies including observing an appropriate immune response in a subject. For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determined empirically.

[0094] An "effective amount" of the composition of the present invention is a dose that elicits antibodies that significantly reduce the likelihood or severity of infection by a microorganism, such as S. pneumoniae, during a subsequent challenge.

[0095] The methods of using the compositions and formulations described herein can be used to prevent and / or reduce primary clinical syndromes caused by microorganisms such as S. pneumoniae, including invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

[0096] Administration of the compositions and formulations described herein may include one or more of the following: injection by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or administration by mucosal administration to the oral / digestive tract, respiratory tract or urogenital tract. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (because nasopharyngeal transport of pneumococci can be more effectively prevented, thus reducing infection at an early stage).

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

[0098] The optimal amounts of the components of a particular vaccine can be determined by standard studies including observing an appropriate immune response in a subject. For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data. In another embodiment, the dosage is determined empirically.

[0099] In one embodiment, the dosage of the aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500 or 700 μg, or 1, 1.2, 1.5, 2, 3, 5 mg or more. In another embodiment, the dosage of the aluminum salt is based on per microgram of recombinant protein.

[0100] Typically, each 0.5 ml dose is formulated to contain: 2 μg of each pneumococcal polysaccharide, but 4 μg of serotype 6B polysaccharide; about 32 μg CRM197 carrier protein (e.g., 32 μg ± 5 μg, ± 3 μg, ± 2 μg or ± 1 μg); 0.125 mg elemental aluminum (0.5 mg aluminum phosphate) adjuvant; sodium chloride and L-histidine buffer. The sodium chloride concentration is about 150 mM (e.g., 150 mM ± 25 mM, ± 20 mM, ± 15 mM, ± 10 mM or ± 5 mM) and about 20 mM (e.g., 20 mM ± 5 mM, ± 2.5 mM, ± 2 mM, ± 1 mM or ± 0.5 mM) L-histidine buffer.

[0101] According to any method of using the compositions or formulations described herein, and in one embodiment, the subject is a human. In certain embodiments, the human patient is an infant (less than 1 year old), a toddler (about 12 to 24 months) or a toddler (about 2 to 5 years old). In other embodiments, the human patient is an elderly patient (> 65 years old). The compositions of the present invention are also suitable for older children, adolescents and adults (e.g., 18 to 45 years old or 18 to 65 years old).

[0102] In one embodiment of the method of using the composition or formulation described herein, the composition or formulation is administered as a single inoculation. In another embodiment, the composition or formulation is administered twice, three times, four times or more, and is fully spaced apart. For example, the composition or formulation can be administered at intervals of 1, 2, 3, 4, 5 or 6 months or any combination thereof. The immunization schedule can follow the schedule specified for the pneumococcal vaccine. For example, the conventional schedule for infants and young children with invasive diseases caused by Streptococcus pneumoniae is 2, 4, 6 and 12-15 months old. Therefore, in a preferred embodiment, the composition is administered in a series of 4 doses at 2, 4, 6 and 12-15 months old.

[0103] The compositions described herein may also include one or more proteins from S. pneumoniae. Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO 02 / 083855 and WO 02 / 053761.

[0104] preparation

[0105] The compositions described herein can be administered by one or more methods known to those skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, and formulated accordingly.

[0106] In one embodiment, the composition described herein is administered by epidermal injection, intramuscular injection, intravenous injection, intraarterial injection, subcutaneous injection or intrarespiratory mucosal injection of liquid preparations. Liquid preparations for injection include solutions and the like.

[0107] The composition can be formulated as a single-dose vial, multi-dose vial, or pre-filled syringe.

[0108] In another embodiment, the composition is administered orally and is therefore formulated into a form suitable for oral administration, i.e., 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.

[0109] 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, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are oils from animal, plant or synthetic sources, such as peanut oil, soybean oil, olive oil, sunflower seed oil, cod liver oil, another marine oil or lipids from milk or eggs.

[0110] The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However, it is generally preferred that the pharmaceutical composition for infusion or injection is substantially isotonic when administered. Therefore, for storage, the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic, it may be diluted to an isotonic solution before administration.

[0111] Isotonic agents can be ionic isotonic agents (e.g., salts) or nonionic isotonic agents (e.g., carbohydrates). Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl2, KCl, and MgCl2. Examples of nonionic isotonic agents include, but are not limited to, sucrose, trehalose, mannitol, sorbitol, and glycerol.

[0112] It is also preferred that at least one pharmaceutically acceptable additive is a buffer. For certain purposes, for example, when the pharmaceutical composition is for infusion or injection, it is generally desirable that the composition comprises a buffer capable of buffering the solution to a pH of 4-10, such as 5-9, such as 6-8.

[0113] The buffer may, for example, be selected from Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate and triethanolamine buffers.

[0114] The buffer may also be selected, for example, from USP compatible buffers for parenteral use, particularly when the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from monoacids such as acetic acid, benzoic acid, gluconic acid, glycerol and lactic acid; diacids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid, polyacids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine and Tris.

[0115] Parenteral vehicles (for subcutaneous, intravenous, intra-arterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Intravenous vehicles include liquid and nutrient supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Examples are sterile liquids, such as water and oils, with or without surfactants and other pharmaceutically acceptable adjuvants. Typically, water, saline, aqueous glucose solutions and related sugar solutions, ethylene glycol (such as propylene glycol or polyethylene glycol), polysorbate 80 (PS-80), polysorbate 20 (PS-20) and poloxamer 188 (P188) are preferred liquid carriers, particularly for injection solutions. Examples of oils are oils of animal, plant or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower seed oil, cod liver oil, another marine oil or lipids from milk or eggs.

[0116] The preparation may also contain a surfactant. Preferred surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tween), especially PS-20 and PS-80; DOWFAX TM Copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trademark POXYL®; octoxynol, the number of repeats of the ethoxy(oxy-1,2-ethanediyl) groups of which may vary, of which octoxynol-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as Tergitol TM NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohol (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); sorbitan esters (commonly known as SPAN), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. The preferred surfactant for inclusion in the emulsion is PS-20 or PS-80.

[0117] Mixtures of surfactants may be used, such as a PS-80 / Span 85 mixture. Combinations of polyoxyethylene sorbitan esters, such as polyoxyethylene sorbitan monooleate (PS-80) and octanols, such as tert-octylphenoxypolyethoxyethanol (Triton X-100), are also suitable. Another useful combination includes laurate 9 plus polyoxyethylene sorbitan esters and / or octanol.

[0118] Preferred amounts of surfactants are: 0.01-1% w / v, especially about 0.1% w / v, for polyoxyethylene sorbitan esters (e.g. PS-80); 0.001-0.1% w / v, especially 0.005-0.02% w / v, for octyl or nonylphenoxypolyoxyethanol (e.g. Triton X-100 or other detergents in the Triton series); 0.1-20% w / v, preferably 0.1-10% w / v, especially 0.1-1% w / v or about 0.5% w / v, for polyoxyethylene ethers (e.g. laurate 9).

[0119] In certain embodiments, the composition consists essentially of L-histidine (20 mM), saline (150 mM) and 0.2% w / v PS-20 (at a pH of 5.8) with 250 μg / mL of APA (aluminum phosphate adjuvant). In simulated manufacturing processes and during transportation using primary packaging, the range of PS-20 can be 0.005% to 0.1% w / v in the presence of PS-20 or PS-80 to control formulation aggregation. The method consists of mixing a blend of up to 44 pneumococcal polysaccharide serotypes in L-histidine, sodium chloride and PS-20, and then mixing this blend with APA and sodium chloride, with or without the addition of an antimicrobial preservative.

[0120] The choice of surfactant may need to be optimized for different drug products and drug substances. For multivalent vaccines containing 15 or more pneumococcal polysaccharide serotypes, PS-20 and P188 are preferred. The choice of chemical method for preparing the conjugate will also affect the stability of the formulation. In particular, as exemplified below, pneumococcal polysaccharide-protein conjugates prepared in aqueous or DMSO solvents and combined into multivalent compositions have significant differences in stability, depending on the specific surfactant system used for the formulation.

[0121] For the formulations described herein, the molecular weight of the poloxamer is typically 1,100Da to 1,7400Da, 7,500Da to 15,000Da, or 7,500Da to 10,000Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of the poloxamer in the formulation of the present invention is 0.001 to 5% w / v, or 0.025 to 1% w / v. The surfactant system containing the poloxamer must also contain a polyol. In some aspects, the polyol is propylene glycol, and the final concentration is 1 to 20% w / v. In some aspects, the polyol is polyethylene glycol 400, and the final concentration is 1 to 20% w / v.

[0122] Suitable polyols for formulation are polymeric polyols, particularly polyether glycols, including but not limited to propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ether. The monomer molecular weight range of propylene glycol is from 425Da to 2,700Da. Polyethylene glycol and polyethylene glycol monomethyl ether with molecular weights of 200Da to 35,000Da can also be obtained, including but not limited to PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEGMME4000. Preferred polyethylene glycol is polyethylene glycol 400. The final concentration of the polyol in the formulation can be 1 to 20% w / v or 6 to 20% w / v.

[0123] The preparation also includes a pH buffered saline solution. The buffer can be, for example, selected from Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, L-histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), MES (2-(N-morpholino) ethanesulfonic acid) and triethanolamine buffer. The buffer can buffer the solution to a pH of 4-10, 5.2-7.5 or 5.8-7.0. In some aspects, the buffer is selected from phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate or citrate. The buffer can also be, for example, selected from the USP compatible buffer for parenteral use, particularly when the pharmaceutical preparation is used for parenteral use. The concentration range of the buffer is 1mM-50mM or 5mM-50mM. In certain aspects, the buffer is L-histidine at a final concentration of 5 mM-50 mM, or succinate at a final concentration of 1 mM-10 mM. In certain aspects, the final concentration of L-histidine is 20 mM ± 2 mM.

[0124] Although saline solutions (i.e., solutions containing NaCl) are preferred, other suitable salts for formulation include, but are not limited to, CaCl2, KCl and MgCl2 and combinations thereof. Nonionic isotonic agents, including but not limited to sucrose, trehalose, mannitol, sorbitol and glycerol may be used in place of salt. Suitable salt ranges include, but are not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline is NaCl, optionally present at a concentration of 20 mM to 170 mM.

[0125] In a preferred embodiment, the formulation comprises an L-histidine buffer containing sodium chloride.

[0126] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, intravenous infusion, transdermal patch, liposome or other modes of administration can be used for administration. In another embodiment, a polymer material is used, for example in a microsphere or in an implant.

[0127] The compositions described herein may also include one or more proteins from S. pneumoniae. Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publication Nos. WO 02 / 083855 and WO 02 / 053761.

[0128] Analytical methods

[0129] Analysis of molecular weight and concentration of conjugates using HPSEC / UV / MALS / RI assay

[0130] The conjugate sample is injected and separated by high performance size exclusion chromatography (HPSEC). Detection is accomplished by ultraviolet (UV), multi-angle light scattering (MALS) and refractive index (RI) detectors in series. Protein concentration is calculated by UV280 using the extinction coefficient. The polysaccharide concentration is deconvoluted from the RI signal (contributed by protein and polysaccharide) using the dn / dc factor (this is the change in the refractive index of the solution, and the change in solute concentration is expressed in mL / g). The average molecular weight of the sample is calculated using the concentration and light scattering information measured on the entire sample peak by Astra software (Wyatt Technology Corporation, Santa Barbara, CA). The molecular weight average of polydisperse molecules has various forms. 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 stated, the term "molecular weight" used throughout the specification is the weight average molecular weight.

[0131] Determination of lysine consumption in conjugated proteins as a measure of the amount of covalent linkage between the polysaccharide and the carrier protein

[0132] Waters AccQ-Tag Amino Acid Analysis (AAA) was used to measure the degree of conjugation in the conjugate samples. The samples were hydrolyzed using gas phase acid hydrolysis in an Eldex workstation to break down the carrier protein into its component amino acids. The free amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized samples were then analyzed using UPLC on a C18 column with UV detection. Average protein concentrations were obtained using representative amino acids other than lysine. The amount of lysine consumed during the conjugation process (i.e., lysine loss) was determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the starting protein.

[0133] Free polysaccharide test

[0134] The free polysaccharide in the conjugate sample (i.e., the polysaccharide not conjugated to CRM197) was determined by first precipitating the free protein and conjugate with deoxycholate (DOC) and hydrochloric acid. The precipitate was then filtered off and the concentration of free polysaccharide in the filtrate was analyzed by HPSEC / UV / MALS / RI. Free polysaccharide was calculated as a percentage of the total polysaccharide measured by HPSEC / UV / MALS / RI.

[0135] Free protein test

[0136] Free polysaccharide, polysaccharide-CRM197 conjugate and free CRM197 in the conjugate samples were separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. Briefly, the samples were mixed with MEKC running buffer containing 25mM borate, 100mM SDS, pH 9.3 and separated in pretreated bare quartz capillaries. The separation was monitored at 200nm and free CRM197 was quantified using a CRM197 standard curve. Free protein results were reported as a percentage of total protein content determined by HPSEC / UV / MALS / RI method.

[0137] Having described various embodiments of the present invention with reference to the accompanying description and drawings, it should be understood that the present invention is not limited to those precise embodiments and that various changes and modifications may be made therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0138] The following examples illustrate but do not limit the invention.

[0139] Example

[0140] Example 1: Preparation of Streptococcus pneumoniae capsular polysaccharide

[0141] 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. EP 0 497 524 B1. The methods described below generally follow the methods described in European Patent No. EP 0 497 524 B1 and are generally applicable to all pneumococcal serotypes unless specifically modified.

[0142] Pneumococcal serotypes 3, 8, 12F isolates were obtained from the University of Pennsylvania (Dr. Robert Austrian). Pneumococcal serotypes 15A, 16F, 23A, 24F, 35B isolates were obtained from Merck Culture Collection. Pneumococcal serotypes 23B and 31 isolates were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). Pneumococcal serotype 17F isolates were obtained from the FDA Office of Biologics (Dr. John Robbins). Pneumococcal serotype 20 isolates were obtained from ATCC. If necessary, specific antisera can be used to distinguish subtypes according to the Quelling reaction. See, for example, U.S. Patent No. 5,847,112. The isolates obtained by further cloning and separation were continuously plated on agar plates in two stages, and the agar plates were composed of a medium without animal components, and the medium contained soy peptone, yeast extract and glucose without hemin. Clonal isolates of each serotype were further expanded in liquid culture using animal component-free medium containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare pre-master cell banks.

[0143] The production of pneumococcal polysaccharides of each serotype consists of cell expansion and batch production fermentation, and then chemical inactivation is performed before downstream purification. The thawed cell bank vials from various serotypes are expanded using a shake flask or culture bottle equipped with a pre-sterilized animal-free growth medium, and the culture medium contains soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate and glucose. The cell expansion culture is grown in a sealed shake flask or culture bottle to minimize gas exchange under temperature and stirring control. After reaching a specified culture density (measured by the optical density of 600nm), a portion of the cell expansion culture is transferred to a production fermenter containing a pre-sterilized animal-free growth medium, and the culture medium contains soy peptone or soy peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate and glucose. Control temperature, pH, pressure and stirring. Airflow coverage is also controlled because bubbling (sparging) is not used.

[0144] When the glucose is almost exhausted, the batch fermentation is terminated by adding the chemical inactivator phenol. Pure phenol is added to a final concentration of 0.8-1.2% to inactivate the cells and release the capsular polysaccharide from the cell wall. Primary inactivation occurs in the fermenter for a specified time, where temperature and agitation are continuously controlled. After primary inactivation, the batch is transferred to another container, where it is maintained at a controlled temperature and agitation for an additional specified time for complete inactivation. This can be confirmed by microbial plating techniques or by verifying the phenol concentration and the specified time. The inactivated broth is then purified.

[0145] Purification of Ps

[0146] The purification of pneumococcal polysaccharides consists of multiple centrifugation, depth filtration, concentration / diafiltration operations and precipitation steps. All steps were performed at room temperature unless otherwise stated.

[0147] The inactivated medium from a fermenter culture of Streptococcus pneumoniae is flocculated with a cationic polymer (e.g., BPA-1000, Petrolite "Tretolite" and "Spectrum 8160" and poly(ethyleneimine), "Millipore pDADMAC"). The cationic polymer binds to impure proteins, nucleic acids and cell debris. After the flocculation step and aging phase, the flocculated solids are removed by centrifugation and multiple depth filtration steps. The clarified medium is concentrated and diafiltered using a 100 kDa to 500 kDa MWCO (molecular weight cutoff) filter. Diafiltration is accomplished using Tris, MgCl2 buffer and sodium phosphate buffer. Diafiltration removes residual nucleic acids and proteins.

[0148] Impurities can be further removed by reprecipitating the polysaccharides in sodium acetate and phenol with denatured alcohol and / or isopropanol. In the phenol precipitation step, sodium acetate and phenol (liquefied phenol or solid phenol) in sodium phosphate saline buffer are loaded into the retentate that has been filtered. Alcohol fractionation of polysaccharides is then performed in two stages. In the first stage, a low percentage of ethanol is added to the preparation to precipitate cell debris and other harmful impurities, while the crude polysaccharides remain in the solution. Impurities are removed via a depth filtration step. Polysaccharides are then recovered from the solution by adding additional isopropanol or denatured alcohol to the batch. The precipitated polysaccharide precipitate is recovered by centrifugation, ground and dried into a powder, and stored frozen at -70°C.

[0149] Example 2: General conjugation method

[0150] Polysaccharide size reduction and oxidation

[0151] Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered at 0.45 micron. Unless otherwise stated, the polysaccharide was homogenized to reduce the polysaccharide molecular weight. The homogenization pressure and the number of passes through the homogenizer were controlled to serotype specific targets (150-1000 bar; 4-7 passes).

[0152] The size-reduced polysaccharide was subjected to 0.2 micron filtration and then concentrated and diafiltered against distilled water using a 5 kDa or 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0153] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation.

[0154] Purified polysaccharides were prepared for conjugation, i.e., activation, using sodium metaperiodate oxidation (see Anderson et al., 1986, J. Immunol. 137: 1181-1186; and U.S. Patent Application Publication No. US20110195086). A 100 mM sodium metaperiodate solution was added to a polysaccharide solution in 50 mM sodium acetate. The amount of sodium metaperiodate added was serotype specific, ranging from about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit to achieve the target level of polysaccharide activation (number of moles of aldehyde per mole of polysaccharide repeating unit). The sample was mixed and protected from light for the target incubation time.

[0155] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 5 kDa or 10 kDa NMWCO tangential flow ultrafiltration membrane, followed by diafiltration against distilled water, and then an additional diafiltration against water. Ultrafiltration for all serotypes was performed at 2-8°C.

[0156] Conjugation

[0157] Purified CRM197 obtained by expression in Pseudomonas fluorescens was diafiltered using a 5 kDa NMWCO tangential flow ultrafiltration membrane with 2 mM phosphate (pH 7.2) buffer and filtered at 0.2 micron as previously described (WO 2012 / 173876 A1).

[0158] The activated polysaccharide was prepared at 1-6 mg Ps / mL and 0.5-30% w / v sucrose concentration for lyophilization. CRM197 was prepared at 6 mg Pr / mL and 1% w / v sucrose concentration for lyophilization.

[0159] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in an equal volume of DMSO. The polysaccharide and CRM197 solutions were mixed to achieve the target polysaccharide concentration and the mass ratio of polysaccharide to CRM197. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate. Sodium cyanoborohydride (1 mol per mol of polysaccharide repeating unit) was added and the conjugation was carried out at 22°C for the target incubation time.

[0160] Reduction with sodium borohydride

[0161] After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. The batch was diluted at about 4°C into 150 mM sodium chloride containing about 0.025% (w / v) polysorbate 20. Potassium phosphate buffer was then added to neutralize the pH.

[0162] Final filtration and product storage

[0163] The conjugate was then dialyzed against 150 mM sodium chloride containing 0.05% (w / v) polysorbate 20 using a 300 kDa NMWC membrane at about 4°C, or diafiltered against 150 mM sodium chloride (with or without 25 mM potassium phosphate pH 7) using a 30 kDa NMWC tangential flow ultrafiltration membrane, and then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride (pH 7.0) containing 0.015% (w / v) polysorbate 20 using a 300 kDa NMWCO tangential flow ultrafiltration membrane at 4°C. The retentate batch was diluted with additional 10 mM histidine in 150 mM sodium chloride (pH 7.0) and filtered at 0.2 microns. The final conjugate solution was distributed into aliquots and frozen at ≤-60°C.

[0164] Example 3: Acid hydrolysis of polysaccharides from serotypes 12F, 23A, 24F and 31

[0165] Conjugation of pneumococcal polysaccharides to proteins by reductive amination in aprotic solvents such as DMSO has been described previously. Typically, the activated polysaccharide (Ps) and protein (Pr) are lyophilized, resuspended in DMSO, and then mixed and incubated with sodium cyanoborohydride and sodium borohydride to achieve conjugation. The polysaccharide can be mechanically reduced in size (e.g., by homogenization) prior to oxidation to reduce the Ps molecular weight and provide a consistent Ps size for conjugation. For many pneumococcal serotypes, conjugation of mechanically reduced and oxidized Ps produces conjugates that meet the target attributes of size, lysine consumption, free polysaccharide, and free protein. However, for some serotypes, it was found that the target conjugation attributes were difficult to achieve using this process even after optimizing the process parameters.

[0166] Reduce Ps size

[0167] Purified pneumococcal capsular polysaccharide powders from serotypes 23A, 24F and 31 were dissolved in water. Different experimental groups were treated mechanically by homogenization or chemically by acid hydrolysis to reduce the molecular weight of Ps. The homogenization pressure and the number of passes through the homogenizer were controlled to serotype specific targets (150-1000 bar; 4-7 passes). Acid hydrolysis was performed by heating the batch to 90-92°C, adding concentrated acetic acid to a final concentration of 200mM, and then incubating for up to 90 minutes. At the end of the incubation period, the batch was neutralized by adding concentrated potassium phosphate pH 7 buffer to a final concentration of 400mM and cooling to ≤22°C. The size-reduced polysaccharides were filtered at 0.2 microns, then concentrated and diafiltered with water using a 5kDa or 10kDa NMWC tangential flow ultrafiltration membrane.

[0168] The polysaccharide was conjugated as described in Example 2.

[0169] The experimental conditions and results are summarized in Table 1.

[0170] Table 1. Summary of experimental groups for Ps size reduction by homogenization compared to acid hydrolysis for S. pneumoniae serotypes 23A, 24F, and 31.

[0171]

[0172] As shown in Table 1, size reduction by acid hydrolysis provides a means to achieve higher lysine loss (serotype 23A), lower free Ps (serotypes 24F and 31), or lower free Pr (serotypes 23A, 24F, and 31) compared to homogenization. For serotypes 23A and 24F, the higher free protein levels in response to homogenization may be associated with aggregated forms, which in turn may lead to higher measured conjugate Mw levels.

[0173] Without being bound by any particular theory, the data suggest that lower molecular weight oxidized polysaccharides (preferably less than 150 KDa) obtained by acid hydrolysis contribute to improved conjugation (less free Ps or free Pr). It is believed that alternative size reduction methods (e.g., by acid hydrolysis) can be used to obtain polysaccharides in this preferred size range to achieve similar conjugation benefits.

[0174] Effect of acid type on acid hydrolysis of serotype 12F at constant pH

[0175] To determine whether the type of acid has an effect on the size of the polysaccharide, acid hydrolysis using hydrochloric acid was compared with acetic acid. Purified pneumococcal capsular polysaccharide powder from S. pneumoniae serotype 12F was dissolved in water and filtered at 0.45 μm. The batch was diluted to 2.5 gPs / L and divided into two arms. The two arms were first heated to 80°C for acid hydrolysis. Acid was added to keep the pH similar between the two arms. Glacial acetic acid was added to one arm to a final concentration of 200 mM and a pH of 2.6. 1 N hydrochloric acid was added to the other arm to a final concentration of 2.5 mM and a pH of 2.7. The two arms were then incubated for 155 minutes. At the end of the incubation period, the arm was neutralized by adding concentrated potassium phosphate pH 7 buffer to a final concentration of approximately 400 mM and cooled to 4°C. The results are shown in Table 2.

[0176] Table 2: Acid hydrolysis of Streptococcus pneumoniae 12F polysaccharide using hydrochloric acid

[0177]

[0178] Both acid hydrolysis conditions produced polysaccharides of similar size.

[0179] Example 4: Effect of the mass ratio of sucrose to polysaccharide on the dissolution of polysaccharides from Streptococcus pneumoniae serotypes 3, 8 and 24F in DMSO

[0180] To prepare polysaccharide conjugates to proteins in aprotic solvents, polysaccharide and protein solutions are typically lyophilized. For most pneumococcal serotypes, activated polysaccharide (Ps) was formulated for lyophilization at 6 mg Ps / mL, sucrose concentration of 5% w / v (50 mg sucrose / mL) in aqueous solution, making the lyophilized material suitable for redissolution in DMSO and conjugation. For serotypes 3, 8 and 24F, this formulation (6 mg Ps / mL, 50 mg sucrose / mL, sucrose:Ps mass ratio = 8.3) was found to produce lyophilized material that was insoluble in DMSO.

[0181] Experiments were conducted to optimize the activated polysaccharide freeze-dried formulation for dissolution in DMSO. In polypropylene containers, activated polysaccharides were prepared between a certain range of polysaccharide concentrations (1-6 mg Ps / mL) and sucrose concentrations (50-300 mg sucrose / mL). The solution was freeze-dried to remove water, then DMSO was added at ambient temperature and mixed to redissolve the polysaccharide. Dissolution was successfully determined by visual observation.

[0182] Tables 3, 4 and 5 show the results for serotypes 3, 24F and 8, respectively.

[0183] Table 3 Lyophilized preparation and dissolution test of serotype 3

[0184]

[0185]

[0186] Table 4 Lyophilized preparation and dissolution test of serotype 24F

[0187]

[0188] *In experimental condition 4, a single small visible particle was observed, which is not believed to be due to incomplete dissolution.

[0189] Table 5 Lyophilized preparation and dissolution test of serotype 8

[0190]

[0191] For these serotypes, complete dissolution was observed in the range of polysaccharide concentrations studied (both lyophilized and dissolved), however dissolution depended on both polysaccharide and sucrose concentrations. Specifically, when the mass ratio of sucrose was less than 30 times the mass ratio of polysaccharide, dissolution in DMSO after lyophilization could not be achieved. When the mass ratio of sucrose was at least 40 times the mass ratio of polysaccharide, the most consistent dissolution results were found.

[0192] As described in Example 2, several groups from the positive lysis conditions were successfully conjugated to CRM197.

[0193] Effects of sugar type and sugar-to-polysaccharide mass ratio on the solubility of polysaccharides from serotype 3 Streptococcus pneumoniae in DMSO

[0194] Experiments were conducted to assess the effects of sugar type and sugar to polysaccharide mass ratio on the dissolved lyophilized activated polysaccharide in DMSO. Activated serotype 3 polysaccharides were prepared in polypropylene containers with 2.5 or 6 mg Ps / mL and a certain range of sugar concentrations (50-150 mg sugar / mL). The sugar tested was sucrose, trehalose and mannitol. The solution was freeze dried to remove water, then DMSO was added at ambient temperature and mixed to redissolve the polysaccharide. Dissolution was successfully determined by visual observation. The results are shown in Table 6.

[0195] Table 6: Effect of sugar type on serotype 3 polysaccharide

[0196]

[0197] * Sticky

[0198] For all sugars used, dissolution in DMSO could be achieved at a sugar mass ratio of 30 or higher, except for mannitol, which seemed to reach its solubility limit in DMSO at 60. The most consistent dissolution results were found when the sugar mass ratio was at least 40 times that of the polysaccharide.

[0199] Another experiment was conducted to investigate the use of combinations of sugars in activated serotype 3 polysaccharide lyophilized formulations. All groups were formulated with a total sugar mass ratio of 40 times, as this ratio produces stable solubility in DMSO. The molecular weight of the polysaccharide was 171 kD. All groups used sucrose alone or with trehalose or with mannitol. The solution was lyophilized to remove water, and then DMSO was added and mixed at ambient temperature to redissolve the polysaccharide. Dissolution success was determined by visual observation. The results are shown in Table 7.

[0200] Table 7 Combinations of sugars used to dissolve polysaccharides in DMSO

[0201]

[0202] Dissolution in DMSO was achieved for all tested groups.

[0203] Example 5: Effect of sodium chloride on conjugation of S. pneumoniae serotypes 15A, 16F, 17F, 20, 24F and 35B using reductive amination in DMSO

[0204] The activated polysaccharide was prepared at 2-6 mg Ps / mL and sucrose concentration of 5-10% w / v in polypropylene containers for lyophilization. CRM197 was prepared at 6 mg Pr / mL and sucrose concentration of 1% w / v for lyophilization.

[0205] The prepared Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in DMSO. For some groups, 5M sodium chloride stock solution was used to spike the CRM197 solution before lyophilization, or in the redissolved Ps solution to reach a final concentration of 10-100mM sodium chloride during the conjugation process. In these experiments, other process parameters remained unchanged.

[0206] The redissolved Ps and CRM197 solutions were blended and mixed to the target concentrations of serotype specific polysaccharides and proteins. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added and conjugated for a serotype specific duration. Reduction with sodium borohydride and final filtration were performed as described in Example 2.

[0207] result

[0208] Figure 1 , 2 Tables 3 and 4 show the results of experiments with polysaccharides from S. pneumoniae serotype 20, demonstrating the effect of sodium chloride on the properties of the conjugate during the conjugation process. Table 8 shows the results of experiments with S. pneumoniae serotypes 16F and 24F.

[0209] like Figure 1 As shown, for serotype 20, increasing the sodium chloride concentration (up to about 50 mM) during the conjugation process increased the conjugate size. Figure 2 As shown, increasing sodium chloride concentration (up to about 25 mM) increases lysine consumption, as Figure 3 As shown, increasing sodium chloride concentration (up to about 50 mM) decreases free Ps and free Pr.

[0210] Table 8 Effect of sodium chloride on the conjugate properties of Streptococcus pneumoniae serotypes 16F and 24F

[0211]

[0212] As shown in Table 8, for serotype 16F, the inclusion of 25 mM sodium chloride during the conjugation process increased the size of the conjugate and the consumption of lysine, while reducing free Ps and free Pr. As shown in Table 8, for serotype 24F, the inclusion of 25 mM sodium chloride during the conjugation process increased the consumption of lysine, while reducing free Ps and free Pr.

[0213] Similar results were found for serotypes 15A and 35B. For serotype 15A, the addition of 25 mM sodium chloride during the conjugation process increased the size of the conjugate and the consumption of lysine, while reducing free polysaccharide and free protein compared to the absence of salt during the conjugation process. For serotype 35B, the addition of 25 mM sodium chloride during the conjugation process increased the size of the conjugate and the consumption of lysine (data not shown), while reducing free protein compared to the absence of salt during the conjugation process.

[0214] Effect of salt type and concentration on the conjugation of Streptococcus pneumoniae serotype 17F using reductive amination in DMSO

[0215] Activated serotype 17F polysaccharide was formulated for lyophilization at 6 mg Ps / mL and sucrose concentration of 5-10% w / v in polypropylene containers. CRM197 was formulated for lyophilization at 6 mg Pr / mL and sucrose concentration of 1% w / v.

[0216] The formulated Ps and CRM197 solutions were lyophilized separately. The lyophilized Ps and CRM197 materials were redissolved in DMSO. For some groups, concentrated salt solutions were spiked into the redissolved Ps solution or Ps-CRM blend to achieve a final concentration of 1-100 mM salt during the conjugation process. The stock solutions used included 1M or 5M sodium chloride, 1M or 3M potassium chloride, and 1M magnesium chloride. In these experiments, other process parameters remained unchanged.

[0217] The redissolved Ps and CRM197 solutions were blended and mixed to 2.7 g Ps / mL and 1.8 mg CRM197 / mL. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added and conjugation was allowed to proceed for 1 hour. After the conjugation reaction, sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. The batch was diluted into 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.

[0218] The conjugate was then dialyzed using a 300 kDa NMWC membrane at approximately 4°C against 150 mM sodium chloride containing 0.05% (w / v) polysorbate 20. The final conjugate solution was dispensed into aliquots and kept at 4°C.

[0219] result

[0220] The results are shown in Table 9. 2% was the detection limit.

[0221]

[0222]

[0223] For sodium chloride and potassium chloride conditions, increasing salt concentration during conjugation increases conjugate size and lysine consumption, and reduces free Ps and free Pr. For both salt types, these effects are stable at about 12.5mM. Compared with salt-free conditions, 25mM and 50mM magnesium chloride show an increase in conjugate size and lysine consumption. However, as the magnesium chloride concentration increases from 25mM to 100mM, free polysaccharide and free protein levels appear to increase, and the degree of conjugation decreases (measured by lysine loss and conjugate size). Therefore, it may be preferred to maintain the concentration of magnesium chloride at 0-50mM.

[0224] Example 6: Formulation of monovalent conjugates

[0225] Pneumococcal polysaccharide-CRM197 conjugates were prepared as described in Examples 2-5. Based on the batch volume and the concentration of the various bulk polysaccharides, the desired volume of the bulk conjugate required to obtain the target concentration of each serotype was calculated. The various serotypes (12F, 15A, 16F, 17F, 23A, 23B, 24F, 31 and 35B) were combined with excipients, sterile filtered and added to APA under mixing conditions. The final concentration of each monovalent conjugate vaccine was 4 μg / mL (w / v PnPs), which contained 20 mM histidine, 150 mM NaCl, 0.2% (w / v) PS-20 and 0.250 mg / mL (w / v Al) in the form of APA.

[0226] Example 7: Immunogenicity study of monovalent conjugates in New Zealand white rabbits (15A, 16F, 17F, 23A, 23B, 24F, 31 and 35B)

[0227] The immunogenicity of the monovalent conjugates was evaluated in the New Zealand white rabbit (NZWR) model. Adult New Zealand white rabbits (NZWR, n=3 / group) were immunized intramuscularly (IM) with 0.25 ml of the corresponding monovalent conjugate vaccine on days 0 and 14 (alternating sides). The monovalent pneumococcal vaccine was administered with 1 μg of PnPs (15A, 16F, 17F, 23A, 23B, 24F, 31 or 35B, each conjugated with CRM197) with 62.5 μg of aluminum phosphate adjuvant (APA) per immunization. Serum was collected before the start of the study (before immunization) and on days 14 (after dosing 1, PD1) and 28 (after dosing 2, PD2). NZWRs were observed at least daily by trained animal care staff for any signs of illness or distress. The vaccine formulation in NZWR was considered safe and well tolerated. All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees of both Merck & Co., Inc (Kenilworth, NJ) and Covance (Denver, PA).

[0228] NZWR serum was tested in an ELISA assay using 1-2 mg / ml of the corresponding PnPs coating concentration to assess the immunogenicity of IgG. Functional antibodies were determined by opsonophagocytosis assay (OPA) according to the previous protocol. See, for example, Caro-Aguilar et al., 2017, Vaccine 35:865-72 and Burton et al., 2006, Clin Vaccine Immunol 13(9):1004-9.

[0229] All monovalent pneumococcal conjugate vaccines were found to be immunogenic in rabbits and produced functional antibodies that killed the corresponding bacterial strains (data not shown). Serotype 12F was found to be immunogenic in mice (data not shown).

Claims

1. A method for freeze-drying a solution comprising a polysaccharide, wherein the polysaccharide is a Streptococcus pneumoniae polysaccharide, wherein the Streptococcus pneumoniae polysaccharide is from serotype 3, 8 or 24F, the method comprising: a) adding a sugar to a solution containing the pneumococcal polysaccharide to obtain a mass ratio of sugar to polysaccharide of 30-50, wherein the sugar is mannitol, sucrose, trehalose or a combination thereof; and b) freeze-drying a solution comprising said Streptococcus pneumoniae polysaccharide.

2. The method according to claim 1, wherein the mass ratio of the sugar to the polysaccharide is 40-50.

3. The method according to claim 1, wherein the sugar concentration is greater than 5%. The method according to claim 1 , wherein the solution comprising polysaccharides further comprises proteins.

5. The method of claim 4, wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM197. The method of claim 1 , further comprising conjugating the polysaccharide to a protein.

7. The method of claim 6, wherein the protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid and CRM197.

8. The method of claim 7, wherein the protein is CRM197.

9. The method of claim 6, wherein the conjugation is by reductive amination.

10. The method of claim 9, wherein the reductive amination is performed under aqueous conditions. The method according to claim 9 , wherein the reductive amination is performed in dimethyl sulfoxide (DMSO).

Images