Enhanced immunogenicity of Streptococcus pneumoniae polysaccharide-protein conjugates

Abstract

The present invention provides immunogenic compositions having one or more polysaccharide-protein conjugates in which one or more polysaccharides derived from the Streptococcus pneumoniae capsule are conjugated to a carrier protein in an aprotic solvent such as dimethyl sulfoxide (DMSO). The present invention also provides a method for providing an enhanced immune response to a pneumococcal polysaccharide-protein conjugate vaccine, comprising administering to a human subject an immunogenic composition comprising a polysaccharide-protein conjugate prepared in DMSO conditions. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present invention provides a method for preparing CRMs using reductive amination in an aprotic solvent such as dimethylsulfoxide (DMSO). 197 The present invention also provides a method for enhancing the immunogenicity of an immunogenic composition comprising at least one Streptococcus pneumoniae polysaccharide conjugated to a carrier protein using reductive amination performed in an aprotic solvent such as DMSO. [Background technology]

[0002] Streptococcus pneumoniae is a Gram-positive bacterium that is the most common cause of invasive bacterial disease in infants and children (pneumonia, bacteremia, meningitis, otitis media, etc.). Pneumococci are encapsulated with chemically bound polysaccharides that confer serotype specificity. There are over 90 known serotypes of pneumococci, and the capsule is the major virulence determinant of pneumococci, not only because it protects the inner surface of the bacterium from complement, but also because it is poorly immunogenic in itself. Polysaccharides are T-cell-independent antigens, so they cannot be processed or presented on MHC molecules to interact with T cells. They can, however, stimulate the immune system through other mechanisms that involve cross-linking of surface receptors on B cells.

[0003] Because children under the age of 2 do not mount an immune response to most polysaccharide vaccines, it has been necessary to render polysaccharides immunogenic by chemical conjugation to protein carriers. Coupling polysaccharides (T-cell-independent antigens) to proteins (T-cell-dependent antigens) confers T-cell-dependent properties to the polysaccharides, including isotype switching, affinity maturation, and memory induction.

[0004] Many conjugation reactions have been used to covalently attach polysaccharides to proteins. Three of the more commonly used methods include: 1) reductive amination: an aldehyde or ketone group on one component of the reaction reacts with an amino or hydrazide group on the other component, and the resulting C=N double bond is subsequently reduced to a C-N single bond by a reducing agent. 2) cyanylation conjugation: the polysaccharide is activated by either cyanogen bromide (CNBr) or 1-cyano-4-dimethylammonium pyridinium tetrafluoroborate (CDAP) to introduce a cyanate group at the hydroxyl group, which upon addition of the protein component forms a covalent bond with the amino or hydrazide group. 3) carbodiimide reaction: a carbodiimide activates a carboxyl group on one component of the conjugation reaction, and the activated carbonyl group reacts with an amino or hydrazide group on the other component. These reactions are also often used to activate the components of the conjugate prior to the conjugation reaction.

[0005] Reductive amination has been utilized to conjugate pneumococcal polysaccharides. See, for example, U.S. Patent No. 8,192,746, U.S. Patent Application Publication No. 20170021006, WO 2011 / 110381, and WO 2015 / 110941. Reductive amination involves two steps: (1) oxidation of an antigen, and (2) reduction of the antigen and carrier protein to form a conjugate. The reduction step can be carried out in an aqueous solvent or an aprotic solvent such as DMSO. See, for example, WO 2016 / 113644. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 8,192,746 [Patent Document 2] U.S. Patent Application Publication No. 20170021006 [Patent Document 3] International Publication No. 2011 / 110381 [Patent Document 4] International Publication No. 2015 / 110941 [Patent Document 5] International Publication No. 2016 / 113644 Summary of the Invention [Means for solving the problem]

[0007] The present invention provides an immunogenic composition comprising a polysaccharide from one or more of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 conjugated to a carrier protein, wherein the conjugation reaction conjugating the polysaccharide to the carrier protein is carried out in an aprotic solvent. In one embodiment, for compositions having the same serotype, one or more serotypes prepared in an aprotic solvent are more immunogenic when compared to the same one or more serotypes prepared under aqueous conditions.

[0008] The present invention provides an immunogenic composition comprising a polysaccharide-protein conjugate prepared from one or more of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 conjugated to a carrier protein, wherein the polysaccharide-protein conjugate is produced by a process comprising the step of conjugating the polysaccharide to the carrier protein in an aprotic solvent.

[0009] The present invention also provides a method of conjugating a polysaccharide from Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F or 38 to a carrier protein, the method comprising the step of conjugating the polysaccharide to the carrier protein in an aprotic solvent.

[0010] The invention also provides a method of treating a subject with an immunogenic composition comprising one or more polysaccharides from Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F or 38 conjugated to a carrier protein, wherein the polysaccharides are conjugated to the carrier protein in an aprotic solvent.

[0011] In certain embodiments, polysaccharides from one or more of S. pneumoniae serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are conjugated to a carrier protein in an aprotic solvent. In certain embodiments, polysaccharides from one or more of S. pneumoniae serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are conjugated to a carrier protein in an aprotic solvent.

[0012] In certain embodiments, polysaccharides from one or more of Streptococcus pneumoniae serotypes 3 and 18C are conjugated to a carrier protein in an aprotic solvent. In one aspect of this embodiment, polysaccharides from Streptococcus pneumoniae serotype 3 are conjugated to a carrier protein in an aprotic solvent. In one aspect of this embodiment, polysaccharides from Streptococcus pneumoniae serotype 18C are conjugated to a carrier protein in an aprotic solvent.

[0013] In certain embodiments, the conjugation reaction used to conjugate the polysaccharide to the carrier protein is reductive amination.

[0014] In certain embodiments, the aprotic solvent is DMSO.

[0015] In certain embodiments, the carrier protein is a CRM 197 It is.

[0016] In certain embodiments, the conjugate prepared in DMSO has a lysine loss value of greater than 5.0. In one aspect, the conjugate prepared in DMSO has a lysine loss value of between 7.0 and 18.0, inclusive.

[0017] In certain embodiments, the immunogenic composition further comprises a polysaccharide from one or more of Streptococcus pneumoniae serotypes 6A, 6B, 7F, 10A, 15B, 19A, 19F, 22F, 23F and 33F conjugated to a carrier protein, and the conjugation reaction conjugating the polysaccharide to the carrier protein is carried out in an aprotic solvent. In certain aspects of this embodiment, the conjugation reaction is reductive amination. In certain aspects, the aprotic solvent is DMSO. In certain aspects, the carrier protein is CRM 197 In one embodiment, the immunogenic composition comprises a polysaccharide from Streptococcus pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F conjugated to a carrier protein, and the conjugation reaction of the polysaccharide from Streptococcus pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F or 23F to the carrier protein is carried out in an aprotic solvent. In a particular embodiment, the polysaccharide is from Streptococcus pneumoniae serotypes 18C, 19A, 19F or 23F.

[0018] In certain embodiments, the immunogenic compositions of the invention comprise polysaccharides from one or more of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 conjugated to a carrier protein. and the conjugation reaction of conjugating the polysaccharide from the Streptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F or 38 to the carrier protein is carried out in an aqueous solvent. In one embodiment, 35-100% of the serotypes in the immunogenic composition are prepared using reductive amination under DMSO conditions and the remaining polysaccharide-protein conjugates are prepared under aqueous conditions.

[0019] In one particular embodiment, the present invention provides a CRM 197 The present invention provides an immunogenic composition consisting essentially of a polysaccharide from Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a polysaccharide, wherein the conjugation reaction of S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F is performed under DMSO conditions and the conjugation reaction of S. pneumoniae serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F is performed in an aqueous solvent, and optionally further comprising about 0.2% w / v PS-20.

[0020] The present invention also provides a method for inducing a protective immune response in a human subject comprising administering any of the immunogenic compositions of the present invention. In certain embodiments, the subject is 50 years of age or older and / or immunocompromised. In certain embodiments, the subject is 2 years of age or younger. In certain embodiments, the subject is immunocompromised.

[0021] The present invention also provides a method for providing an enhanced immune response to a pneumococcal polysaccharide (PnP) protein conjugate vaccine, comprising administering to an animal subject an immunogenic composition comprising a polysaccharide-protein conjugate comprising pneumococcal capsular polysaccharides from a first set of two or more Streptococcus pneumoniae serotypes conjugated to one or more carrier proteins, wherein two or more of the polysaccharide-protein conjugates from the first set are prepared using reductive amination under DMSO conditions. In one embodiment, the enhanced immune response is relative to a control animal receiving an immunogenic composition in which one or more of the two or more polysaccharide-protein conjugates from the first set are prepared using reductive amination under aqueous conditions. In one embodiment, the control animal is a mouse. In another embodiment, the control animal is a human. In a particular embodiment, the method uses a pneumococcal polysaccharide protein conjugate vaccine comprising a further polysaccharide-protein conjugate comprising pneumococcal capsular polysaccharides from a second set of pneumococcal serotypes conjugated to one or more carrier proteins, the polysaccharide-protein conjugates being prepared using reductive amination under aqueous conditions, the serotypes from the second set being different from the serotypes of the first set.

[0022] In certain embodiments, the method uses a pneumococcal polysaccharide protein conjugate vaccine wherein the pneumococcal serotype is selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 33F, 34, 35A, 35B, 35F and 38.

[0023] In a particular embodiment, the method uses a pneumococcal polysaccharide-protein conjugate vaccine in which polysaccharide-protein conjugates derived from serotype 3 or 18C are prepared using reductive amination under DMSO conditions.

[0024] In a particular embodiment, the method uses a pneumococcal polysaccharide-protein conjugate vaccine, wherein the polysaccharide-protein conjugate from a first set of pneumococcal serotypes is selected from serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F.

[0025] In a particular embodiment, the method uses a pneumococcal polysaccharide-protein conjugate vaccine in which polysaccharide-protein conjugates from a first set of pneumococcal serotypes include serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F are prepared using reductive amination under DMSO conditions, and polysaccharide-protein conjugates from a second set of serotypes are prepared under aqueous conditions.

[0026] In one particular embodiment, the method uses pneumococcal polysaccharide-protein conjugate vaccines in which polysaccharide-protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F are prepared using reductive amination under DMSO conditions, and polysaccharide-protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F are prepared under aqueous conditions.

[0027] In certain embodiments, the method uses a pneumococcal polysaccharide-protein conjugate vaccine in which polysaccharide-protein conjugates from 35-100% of the serotypes are prepared using reductive amination under DMSO conditions and the remaining polysaccharide-protein conjugates are prepared under aqueous conditions. In one embodiment, polysaccharide-protein conjugates from 45-80% of the serotypes are prepared using reductive amination under DMSO conditions and the remaining polysaccharide-protein conjugates are prepared under aqueous conditions. In another embodiment, polysaccharide-protein conjugates from 75-100% of the serotypes are prepared using reductive amination under DMSO conditions and the remaining polysaccharide-protein conjugates are prepared under aqueous conditions.

[0028] In certain embodiments, the method further comprises the step of: providing a carrier protein comprising Neisseria meningitides outer membrane protein complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM 197 In one embodiment, a pneumococcal polysaccharide protein conjugate vaccine is used, the carrier protein being selected from the group consisting of: 197 It is.

[0029] In certain embodiments, the method employs a pneumococcal polysaccharide protein conjugate vaccine having a higher percentage of formed glycopeptide bonds as measured by a protein lysine loss value of greater than 5.0 when the conjugate is prepared using reductive amination under DMSO conditions. In one aspect of this embodiment, the conjugate prepared using reductive amination under DMSO conditions has a lysine loss value of 7.0 to 18, inclusive. In another aspect, the conjugate prepared using reductive amination under DMSO conditions has a lysine loss value of greater than 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or 10.0.

[0030] In another specific embodiment, the present invention provides a CRM 197The present invention provides a method for providing an enhanced immune response to a pneumococcal polysaccharide (PnP) protein conjugate vaccine consisting essentially of polysaccharides from pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to a polysaccharide, the method comprising administering to a human subject an immunogenic composition comprising polysaccharide-protein conjugates from a first set and a second set of pneumococcal serotypes, the first set of serotypes consisting of 6A, 6B, 7F, 18C, 19A, 19F and 23F and prepared using reductive amination under DMSO conditions, and the second set of serotypes consisting of 1, 3, 4, 5, 9V, 14, 22F and 33F and prepared under aqueous conditions.

[0031] In a particular embodiment, the enhanced immune response in animals vaccinated with the immunogenic composition produced by the method of the present invention is measured by the geometric mean titer of serum IgG or opsonophagocytic antibodies. In one aspect, the enhanced immune response against a pneumococcal serotype is 10% or more compared to a polysaccharide-protein conjugate from the same pneumococcal serotype prepared under aqueous conditions. In one embodiment, the animal is a mouse. In another embodiment, the animal is a human.

[0032] In certain embodiments, the method is used on a human subject over the age of 50. In certain embodiments, the method is used on a human subject under the age of 2. In certain embodiments, the method is used on an immunocompromised human subject.

[0033] The present invention also provides a method for preparing a pneumococcal polysaccharide-protein conjugate by reductive amination, comprising the steps of: a) reacting a pneumococcal polysaccharide selected from serotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F with an amount of an oxidizing agent (e.g., periodate) to form an activated polysaccharide having an activation level of 0.05 to 0.22; b) reacting the activated polysaccharide with a carrier protein in an aprotic solvent, optionally in the presence of a reducing agent, to form a polysaccharide-protein conjugate; The conjugate has a lysine loss value within the range of 7.0 to 18.0, inclusive.

[0034] In certain embodiments, the activation level is between 0.09 and 0.22.

[0035] In certain embodiments, the oxidizing agent is periodate.

[0036] In certain embodiments, the activation level is measured by derivatizing aldehydes on the polysaccharide with thiosemicarbazide.

[0037] In certain embodiments, the reducing agent is a cyanoborohydride salt, such as sodium cyanoborohydride.

[0038] In certain embodiments, the carrier protein is tetanus toxoid, diphtheria toxoid, and CRM 197 In one embodiment, the carrier protein is selected from the group consisting of CRM 197 It is.

[0039] The present invention also provides a quantitative method for determining aldehyde levels (i.e., periodate activation levels) in an activated polysaccharide, comprising: a) derivatizing the activated polysaccharide by reacting with a derivatizing agent until completion (i.e., the reaction plateau) to form a derivatized polysaccharide; b) isolating the derivatized polysaccharide by high performance size exclusion chromatography (to remove unreacted derivatizing agent and matrix components); and c) Quantifying the UV absorbance of the derivatized polysaccharide The present invention provides a method comprising:

[0040] The derivatizing agent can be selected from the group consisting of thiosemicarbazide, thiosemicarbazide structural analogs, hydrazides, hydrazines, semicarbazides, semicarbazide structural analogs, aminooxy compounds, or aromatic amines.

[0041] In one embodiment, quantification in step c) is by comparison to a derivative standard. In one embodiment, quantification in step c) is by measurement against a predefined extinction coefficient. [Brief description of the drawings]

[0042] [Figure 1] Extent of conjugation at different lysine sites on CRM197 as determined by tryptic peptide mapping. Serotype 19 APs-CRM197 conjugates prepared by reductive amination in aqueous solution or DMSO were digested with trypsin and analyzed by LC-UV-MS. Loss of peptide signal compared to CRM197 control samples was plotted against the conjugation site. [Diagram 2] Electrochemiluminescence (ECL) immunogenicity results from mouse test groups comparing serotype 3Ps-CRM197 conjugates prepared by reductive amination in aqueous solution or in DMSO, and conjugates formulated with aluminum phosphate adjuvant (APA). Results are shown pre-vaccination (Pre) and post-3 dose (PD3). Results for the APA only control are also shown. [Diagram 3] Phagocytic activity (OPA) results after the third dose from mouse test groups comparing serotype 3Ps-CRM197 conjugates prepared by reductive amination in aqueous solution or in DMSO. OPA results for pre-vaccination (pre-immune) and APA only controls are also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention provides immunogenic compositions comprising pneumococcal polysaccharide-protein conjugates, the conjugates being prepared using reductive amination in an aprotic solvent such as DMSO, from at least one pneumococcal serotype. The invention is based in part on the discovery that DMSO used as a solvent during reductive amination of polysaccharide-protein conjugates unexpectedly results in superior stability and enhanced immunogenicity for those serotypes compared to the same conjugates prepared under aqueous conditions. The invention relates to the advantage of DMSO solvent in enhancing the covalent association of polysaccharide to protein by direct consumption of lysine residues on the surface of the carrier protein. For most serotypes tested, a "lysine loss" that gives good immunogenicity (≧7.0) could be achieved with lower polysaccharide activation levels (0.05-0.22) by conjugation in aprotic solvents than in aqueous buffers. The increased covalent association has direct benefit in increasing the stability of polysaccharide-protein conjugates and enhancing the immune response to those particular polysaccharide antigens conjugated in DMSO.

[0044] Without being bound by any theory, one possible mechanism for the enhanced immunogenicity observed with glycoconjugates prepared in DMSO is that an increased number of linkages between lysine residues on the surface of the carrier protein and the carbohydrate (capsular polysaccharide) increases the number of attachment points between the protein and the polysaccharide, thereby conferring stability and preventing the destruction or chemical depolymerization of the peptide-carbohydrate bond. See, e.g., Hsieh, Characterization of Saccharide-CRM 197See Conjugate Vaccines in Brown F, Corbel M, Griffiths E (eds): Physico-Chemical Procedures for the Characterization of Vaccines. Dev. Biol. Basel, Karger, 2000, vol. 103, pp. 93-104. An additional benefit of the increased polysaccharide-protein linkages created during conjugation in DMSO solvent may be an increased likelihood of successful presentation of peptide-carbohydrates to T cells. Due to genetic variability in human populations resulting in variable sensitivity and ability to associate with or load specific peptide sequences conjugated to carbohydrate antigens, the increased number of attachment points on the carrier protein may increase the likelihood of successful antigen presentation on the APC surface, allowing T cell-dependent responses to alternative T cell-independent antigens. Another possible mechanism for the observed increased immunogenicity with conjugation in DMSO solvent is the increased affinity of the CRM in organic solvents. 197 This may be due to denaturation, which renders additional lysines available for polysaccharide attachment, increasing the likelihood of glycopeptide presentation on the surface of APCs and resulting in T cell-dependent responses to distinct peptide epitopes. See Avci et al., 2011, Nature Medicine 17:1602-1610.

[0045] Modified CRM during conjugation 197 Yet another advantage of conjugation in organic solvents to produce 197 There may be a reduction in the immunological interference of antibodies against the epitope. An additional benefit of the increased polysaccharide-protein binding created during conjugation in DMSO solvent may be that larger sized polysaccharide-protein conjugates are formed, resulting in enhanced immunogenicity. It is believed that the compositions of the present invention provide a significant advantage in inducing a human response.

[0046] As shown in Example 5, polysaccharide-protein conjugates prepared from S. pneumoniae serotype 3 using reductive amination in DMSO showed increased immunogenicity in a mouse model as measured by phagocytic activity (OPA) (compared to the same conjugates prepared using reductive amination in water). Furthermore, as shown in Example 6, a 15-valent pneumococcal conjugate vaccine with seven serotypes prepared using reductive amination in DMSO (and eight other serotypes prepared in aqueous solvent) tended to show superior immunogenicity in humans (with four serotypes superior with statistical significance) for all seven serotypes prepared in DMSO compared to the corresponding 15-valent PCV when all 15 serotypes were prepared in aqueous solvent.

[0047] As shown in Example 4, the CRM in the serotype 19A conjugate (compared to the CRM197 control) 197 Plotting peptide signal reduction for lysine positions on the protein against potential conjugation sites revealed additional conjugation sites for previously identified common human T cell peptide epitopes (see Raju et al., 1995, Eur. J. Immunol. 25:3207-3214; CRM 197 (Peptides 411-430 and 431-450 of the sequences). Thus, in certain embodiments, the present invention also provides one or more polysaccharide-CRM 197 An immunogenic composition comprising a conjugate, the polysaccharide-CRM 197 At least one of the conjugates is prepared in an aprotic solvent, and the conjugate prepared in the aprotic solvent exhibits a higher CRM than the same conjugate prepared in an aqueous solvent. 197 The present invention also relates to immunogenic compositions that demonstrate greater accessibility of lysine residues at amino acids 411-430 or 431-450 of one or more polysaccharide-CRMs. 197 An immunogenic composition comprising a conjugate, the polysaccharide-CRM 197At least one of the conjugates is prepared in an aprotic solvent, and the CRM in the conjugate prepared in the aprotic solvent 197 In a particular embodiment, the present invention relates to an immunogenic composition comprising a polysaccharide in an aprotic solvent, the polysaccharide comprising one or more lysine residues in the CRM 197 CRM, including conjugation to 197 Especially CRM 197 In a particular aspect of this embodiment, the CRM in a conjugate prepared in an aprotic solvent is 197 of is conjugated at greater than 10%. In these embodiments, the polysaccharide may be from any organism suitable for preparing an immunogenic composition. In particular aspects, the polysaccharide is from Neisseria meningitidis or Streptococcus pneumoniae. The polysaccharide may be from any serotype of these organisms.

[0048] As used herein, the term "aqueous solvent" or "aqueous conditions" when used in conjunction with conjugation, such as reductive amination, refers to the use of water as the solvent for the conjugation reaction. Except for the absence of organic solvents, the water may contain buffers and other components.

[0049] As used herein, the term "aprotic solvent" when used with conjugation such as reductive amination refers to the use of a polar aprotic solvent or a combination of polar aprotic solvents as a solvent for the conjugation reaction. Examples of polar aprotic solvents include, but are not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF) and hexamethylphosphoramide (HMPA). Some water may be present in the aprotic solvent, for example, up to 1%, 2%, 5%, 10% or 20%.

[0050] As used herein, "DMSO solvent" and "DMSO conditions" are used interchangeably.

[0051] As used herein, the term "comprises", when used in reference to the immunogenic compositions of the invention, refers to the inclusion of any other components such as adjuvants and excipients (subject to the limitations of the phrase "consisting of" for antigen mixtures). The term "consisting of", when used in reference to a mixture of multivalent polysaccharide-protein conjugates, refers to the mixture having those particular pneumococcal polysaccharide protein conjugates, but not other pneumococcal polysaccharide protein conjugates derived from different serotypes.

[0052] As used herein, "lysine loss" refers to the amount of lysine consumed during conjugation and is determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the starting protein. Example 4 describes one method for determining "lysine loss".

[0053] As used herein, the term "polysaccharide" is meant to encompass any antigenic sugar element (or antigenic unit) commonly used in the field of immunological and bacterial vaccines, including, but not limited to, "saccharide," "oligosaccharide," "polysaccharide," "liposaccharide," "lipo-oligosaccharide (LOS)," "lipopolysaccharide (LPS)," "glycosylates," "conjugate glycosides," and the like.

[0054] When referring to the percentage of serotypes in an immunogenic composition prepared in an aprotic solvent (e.g., DMSO) and the remaining polysaccharide-protein conjugate prepared under aqueous conditions, it is simply meant to refer to the number of serotypes prepared in the aprotic solvent divided by the total number of serotypes in the composition.

[0055] As used herein, all ranges of pH, temperature, concentration, etc. are meant to include both end values. For example, a pH range of 5.0 to 9.0 is meant to include a pH of 5.0 and a pH of 9.0. Similarly, a temperature range of 4 to 25°C is meant to include both the outer limits of the range, i.e., 4°C and 25°C.

[0056] polysaccharide The pneumococcal capsular polysaccharides that can be prepared by the method of the invention, i.e. reductive amination in an aprotic solvent, include, but are not limited to, the serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38. The polysaccharides may be used in the form of oligosaccharides. These are conveniently formed by fragmentation (e.g. by hydrolysis) of purified polysaccharides, usually followed by purification of fragments of the desired size.

[0057] In certain embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 are prepared using reductive amination in an aprotic solvent. In certain aspects, pneumococcal polysaccharides from one or more of serotypes 1, 3, 4, 5, 9V, 11A, 12F and 14 are prepared using reductive amination in an aprotic solvent. In certain embodiments, pneumococcal polysaccharides from one or more of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 are prepared using reductive amination in an aprotic solvent. In certain embodiments, pneumococcal polysaccharides from one or both of serotypes 3 or 18C are conjugated to a carrier protein using reductive amination in an aprotic solvent. Polysaccharides from other serotypes in the multivalent composition may be conjugated using reductive amination in an aqueous or aprotic solvent. Polysaccharides from other serotypes in the multivalent composition may also be conjugated using other chemistries that may be performed in an aprotic or aqueous solvent.

[0058] Capsular polysaccharides from Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and sized to a certain size by known methods (see, for example, EP 497524 and EP 497525), and preferably by microfluidization, accomplished using a homogenizer, or by chemical hydrolysis. In one embodiment, each pneumococcal polysaccharide serotype is grown in a soy-based medium. The individual polysaccharides are then purified by standard steps including centrifugation, precipitation and ultrafiltration. See, for example, US Patent Publication No. 2008 / 0286838 and US Patent No. 5,847,112. Polysaccharides can be sized to reduce viscosity in polysaccharide samples and / or to improve filterability of conjugated products using techniques such as mechanical or chemical sizing. Chemical hydrolysis can be performed using acetic acid. Mechanical sizing can be performed using high pressure homogenizing shear.

[0059] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of 5 kDa to 4,000 kDa. The molecular weight can be calculated by size exclusion chromatography (SEC) in combination with a multi-angle light scattering detector (MALS) and a refractive index detector (RI). In other such embodiments, the polysaccharide has a molecular weight of 10 kDa to 4,000 kDa; 50 kDa to 4,000 kDa; 50 kDa to 3,000 kDa; 50 kDa to 2,000 kDa; 50 kDa to 1,500 kDa; 50 kDa to 1,000 kDa; 50 kDa to 750 kDa; 50 kDa to 500 kDa; 100 kDa to 4,000 kDa; 100 kDa to 3,000 kDa; 100 kDa to 2,000 kDa. Da; 100kDa to 1,500kDa; 100kDa to 1,000kDa; 100kDa to 750kDa; 100kDa to 500kDa; 100 to 400kDa; 200kDa to 4,000kDa; 200kDa to 3,000kDa; 200kDa to 2,000kDa; 200kDa to 1,500kDa; 200kDa to 1,000kDa, or 200kDa to 500kDa.

[0060] The purified polysaccharides can be chemically activated to allow the saccharides to react with a carrier protein. The purified polysaccharides can be coupled to a linker. Once activated or coupled to a linker, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugates may be prepared by known coupling techniques.

[0061] The polysaccharide can be coupled to the linker to form a polysaccharide-linker intermediate in which the free end of the linker is an ester group. Thus, the linker is one in which at least one end is an ester group. The other end is selected so that the linker can react with the polysaccharide to form the polysaccharide-linker intermediate.

[0062] The polysaccharide can be coupled to the linker using the primary amine groups therein. In this case, the linker typically has ester groups at both ends. This allows coupling to occur by reacting one of the ester groups with a primary amine group in the polysaccharide by nucleophilic acyl substitution. The reaction results in a polysaccharide-linker intermediate in which the polysaccharide is coupled to the linker via an amide bond. The linker is thus a bifunctional linker that provides a first ester group for reaction with the primary amine groups in the polysaccharide and a second ester group for reaction with the primary amine groups in the carrier molecule. A typical linker is adipic acid N-hydroxysuccinimide diester (SIDEA).

[0063] Coupling can also occur indirectly, ie, by means of an additional linker used to derivatize the polysaccharide prior to coupling to the linker.

[0064] The polysaccharide is coupled to the further linker using the carbonyl group at the reducing end of the polysaccharide. This coupling comprises two steps: (a1) reacting the carbonyl group with the further linker and (a2) reacting the free end of the further linker with the linker. In these embodiments, the further linker typically has primary amine groups at both ends, which allows step (a1) to be carried out by reacting one of the primary amine groups with a carbonyl group in the polysaccharide by reductive amination. A primary amine group that is reactive with the carbonyl group in the polysaccharide is used. A hydrazide or hydroxylamino group is suitable. The same primary amine group is typically present at both ends of the further linker. The reaction results in a polysaccharide-further linker intermediate in which the polysaccharide is coupled to the further linker via a C-N bond.

[0065] The polysaccharide can be coupled to the further linker using different groups therein, in particular carboxyl groups. This coupling involves two steps: (a1) reacting said groups with the further linker and (a2) reacting the free end of the further linker with the linker. In this case, the further linker typically has primary amine groups at both ends, which allows step (a1) to be carried out by reacting one of the primary amine groups with a carboxyl group in the polysaccharide by EDAC activation. A primary amine group that is reactive with the EDAC-activated carboxyl group in the polysaccharide is used. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the further linker. The reaction results in a polysaccharide-further linker intermediate in which the polysaccharide is coupled to the further linker via an amide bond.

[0066] In one embodiment, chemical activation of the polysaccharide and subsequent conjugation to the carrier protein by reductive amination can be accomplished by means described in U.S. Patent Nos. 4,365,170, 4,673,574 and 4,902,506, U.S. Patent Publication Nos. 2006 / 0228380, 2007 / 184072, 2007 / 0231340 and 2007 / 0184071, and WO 2006 / 110381, 2008 / 079653 and 2008 / 143709. The chemistry may require activation of the pneumococcal polysaccharide by reaction with any oxidizing agent that oxidizes the terminal hydroxyl groups to aldehydes, such as periodate salts (including sodium periodate, potassium periodate or periodic acid). The reaction results in random oxidative cleavage of vicinal hydroxyl groups on carbohydrates with the formation of reactive aldehyde groups.

[0067] In one embodiment, the polysaccharide is reacted with 0.01-10.0, 0.05-5.0, 0.1-1.0, 0.5-1.0, 0.7-0.8, 0.05-0.5, 0.1-0.3 molar equivalents of oxidizing agent. In one embodiment, the polysaccharide is reacted with about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 molar equivalents of oxidizing agent. To achieve limited polysaccharide activation, it is generally preferred to use smaller amounts of periodate for activation, e.g., 0.1-0.3 Meq (e.g., 0.05-0.22 or 0.09-0.22 moles of aldehyde per mole of polysaccharide repeating unit). As used herein, "activation level" refers to the number of moles of aldehyde per mole of polysaccharide repeating unit. The less activated the polysaccharide, the more native the polysaccharide will be, i.e., fewer hydroxyl groups are converted to aldehydes.

[0068] In another embodiment, the duration of the oxidation reaction is between 1 hour and 50 hours, between 10 hours and 30 hours, between 15 hours and 20 hours, between 15 hours and 17 hours, or about 16 hours.

[0069] In another embodiment, the temperature of the oxidation reaction is maintained between 15° C. and 45° C., between 15° C. and 30° C., between 20° C. and 25° C. In another embodiment, the temperature of the reaction is maintained at about 23° C.

[0070] Coupling to carrier proteins is carried out by reductive amination, by direct amination of the lysyl groups of the protein. For example, conjugation is carried out by reacting a mixture of activated polysaccharide and carrier protein with a reducing agent, such as sodium cyanoborohydride, in the presence of nickel. The conjugation reaction can take place in aqueous solution or in the presence of dimethylsulfoxide (DMSO). See, for example, US Patent Publication Nos. 2015 / 0231270 and 2011 / 0195086 and European Patent No. EP0471177B1. Unreacted aldehydes are then capped by the addition of a strong reducing agent, such as sodium borohydride.

[0071] 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 covalent bond. The polysaccharide may be reduced in size prior to oxidation. Mechanical methods (e.g., homogenization) or chemical hydrolysis may be used. Chemical hydrolysis may be carried out with acetic acid. The oxidation step may involve reaction with periodate. For the purposes of the present invention, the term "periodate" includes both periodate and periodic acid. The term also includes metaperiodate (IO 4 - ) and orthoperiodate (IO 6 - ), including various salts of periodate (e.g., sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is lyophilized in the presence of metaperiodate, preferably sodium periodate (NaIO 4 In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

[0072] In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound (as described in WO 2014 / 097099), in the presence of an oxidizing agent that selectively oxidizes primary hydroxyls. In the reaction, the actual oxidizing agent is an N-oxoammonium salt in the catalytic cycle. In one embodiment, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one embodiment, the stable nitroxyl or nitroxide radical compound has a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one embodiment, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one embodiment, the oxidizing agent is a molecule having an N-halo moiety. In one embodiment, the oxidizing agent is selected from the group consisting of 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.

[0073] In a particular embodiment, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-chlorosuccinimide (NCS) as co-oxidizing agents (as described in WO 2014 / 097099). Thus, in one embodiment, glycoconjugates from Streptococcus pneumoniae are obtained by a method comprising the steps of a) reacting a sugar with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous medium to produce an activated sugar, and b) reacting the activated sugar with a carrier protein containing one or more amine groups (hereinafter said method is referred to as "TEMPO / NCS-reductive amination").

[0074] The oxidation reaction may be quenched by the addition of a quenching agent, which may be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites or phosphorous acids (such as glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, ascorbic acid, etc.).

[0075] The second step of the conjugation process is to reduce the imine (Schiff base) bond between the activated polysaccharide and the carrier protein to form a stable conjugate bond (so-called reductive amination) using a reducing agent. Suitable reducing agents include cyanoborohydride (such as sodium cyanoborohydride or sodium borohydride). In one embodiment, the reducing agent is sodium cyanoborohydride.

[0076] In a particular embodiment of the method 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 DMSO or DMF (dimethylformamide) solvent. If lyophilized, the activated polysaccharide and carrier protein can be reconstituted using DMSO or DMF solvent. In one embodiment, the aprotic solvent is DMSO.

[0077] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH 4 Suitable alternatives include sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BH 3 , benzylamine-BH 3 or amine boranes such as 5-ethyl-2-methylpyridine borane (PEMB), or borohydride exchange resins. Following conjugation (reduction reaction and optional capping), the glycoconjugates may be purified (concentrated with respect to the amount of polysaccharide-protein conjugates) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration, precipitation / elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography), and depth filtration. In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange or size exclusion chromatography.

[0078] Glycoconjugates prepared using reductive amination in aprotic solvents are commonly used in multivalent pneumococcal conjugate vaccines. Thus, in certain embodiments for multivalent compositions where not all serotypes are prepared in aprotic solvents, the reduction reaction of the remaining serotypes can be carried out in aqueous solvents (e.g., pH 6.0-8.5, 7.0-8.0, or 7.0-7.5) using 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 The reaction is carried out in a solvent selected from N-(2-hydroxyethyl)piperazine-N-(2-hydroxypropanesulfonic 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), bicine or HEPB.

[0079] In some embodiments, the glycoconjugates of the present invention comprise a polysaccharide having a molecular weight between 10 kDa and 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 25 kDa and 5,000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 50 kDa and 1,000 kDa. In other such embodiments, the polysaccharide has a molecular weight between 70 kDa and 900 kDa. In other such embodiments, the polysaccharide has a molecular weight between 100 kDa and 800 kDa. In other such embodiments, the polysaccharide has a molecular weight between 200 kDa and 600 kDa. In further such embodiments, the polysaccharide has a molecular weight of from 100 kDa to 1000 kDa; from 100 kDa to 900 kDa; from 100 kDa to 800 kDa; from 100 kDa to 700 kDa; from 100 kDa to 600 kDa; from 100 kDa to 500 kDa; from 100 kDa to 400 kDa; from 100 kDa to 300 kDa; from 150 kDa to 1,000 kDa; from 150 kDa to 900 kDa; a~800kDa;150kDa~700kDa;150kDa~600kDa;150kDa~500kDa;150kDa~400kDa;150kDa~300kDa;200k Da~1,000kDa;200kDa~900kDa;200kDa~800kDa;200kDa~700kDa;200kDa~600kDa;200kDa~500kDa;2 00kDa~400kDa;200kDa~300;250kDa~1,000kDa;250kDa~900kDa;250kDa~800kDa;250kDa~700kDa;2 50kDa~600kDa;250kDa~500kDa;250kDa~400kDa;250kDa~350kDa;300kDa~1,000kDa;300kDa~900kDa a;has a molecular weight of 300kDa~800kDa;300kDa~700kDa;300kDa~600kDa;300kDa~500kDa;300kDa~400kDa;400kDa~1,000kDa;400kDa~900kDa;400kDa~800kDa;400kDa~700kDa;400kDa~600kDa;500kDa~600kDa.

[0080] In some embodiments, the glycoconjugates of the present invention have a molecular weight between 1,000 kDa and 10,000 kDa. In other such embodiments, the polysaccharides have a molecular weight between 1,000 kDa and 7,000 kDa. In other such embodiments, the polysaccharides have a molecular weight between 1,000 kDa and 6,000 kDa.

[0081] In certain embodiments, the conjugation reaction is carried out by reductive amination, where nickel is used for greater conjugation reaction efficiency and to help remove free cyanide.Transition metals are known to form stable complexes with cyanide, and are known to improve the reductive methylation of protein amino groups and formaldehyde using sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203: 331-334; Jentoft et al. Anal Biochem. 1980, 106: 186-190).By complexing the remaining inhibitory cyanide, the addition of nickel increases the consumption of protein during conjugation, leading to the formation of larger and potentially more immunogenic conjugates.

[0082] Differences in starting cyanide levels among sodium cyanoborohydride reagent lots can also lead to variability in conjugation performance, resulting in variations in conjugate size and Ps vs. CRM of the conjugate. 197 The addition of nickel reduced conjugation variability by complexing the cyanide, eliminating differences between lots of sodium cyanoborohydride.

[0083] A suitable alternative chemistry involves activating the sugar with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Thus, the activated sugar can be coupled directly or via a spacer (linker) group to an amino group on a carrier protein. For example, the spacer can be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., with GMBS) or a haloacetylated carrier protein (e.g., with iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB or SIA or SBAP). For example, a cyanate ester (which may have been created by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is conjugated to the carrier protein via a carboxyl group on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described in WO 93 / 15760, WO 95 / 08348 and WO 96 / 29094, as well as Chu et al., 1983, Infect. Immunity 40:245-256.

[0084] Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98 / 42721. Conjugation may involve a carbonyl linker, which may be formed by reacting the free hydroxyl group 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) and then reacting with the protein to form a carbamate bond. This may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling of the CDI carbamate intermediate to an amino group on the protein.

[0085] After the capsular polysaccharide is conjugated to the carrier protein, the polysaccharide-protein conjugate is purified (concentrated with respect to the amount of polysaccharide-protein conjugate) by one or more of a variety of techniques. Examples of these techniques are well known to those skilled in the art and include concentration / diafiltration operations, ultrafiltration, precipitation / elution, column chromatography and depth filtration. See, for example, U.S. Patent No. 6,146,902.

[0086] Another method for characterizing the glycoconjugates of the invention is to characterize the carrier protein (e.g., CRMP) that becomes conjugated to the saccharide. 197) which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation reduces the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In one embodiment, the degree of conjugation of the glycoconjugates of the invention is 2-18, 2-13, 2-10, 2-8, 2-6, 2-5, 2-4, 3-15, 3-13, 3-10, 3-8, 3-6, 3-5, 3-4, 5-18, 5-13, 7-18, 7-13, 8-18, 8-13, 10-18 or 10-13. 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 one embodiment, the degree of conjugation of the glycoconjugates of the invention is 7 to 18. In some such embodiments, the carrier protein is a CRM 197 It is.

[0087] The glycoconjugates of the invention can also be characterized by the ratio of saccharide to carrier protein (w / w). In some embodiments, the ratio of polysaccharide to carrier protein (w / w) in the glycoconjugate is 0.5 to 3.0 (such as 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 saccharide to carrier protein (w / w) is 0.5-2.0, 0.5-1.5, 0.8-1.2, 0.5-1.0, 1.0-1.5 or 1.0-2.0. In further embodiments, the ratio of saccharide to carrier protein (w / w) is 0.8-1.2. In one embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is 0.9-1.1. In some such aspects, the carrier protein is a CRM. 197The glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently bound to a carrier protein, but are still present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound, adsorbed, or entrapped within or with it).

[0088] In one embodiment, the glycoconjugate comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides compared to the total amount of polysaccharides. In one embodiment, the glycoconjugate comprises less than about 25% free polysaccharides compared to the total amount of polysaccharides. In one embodiment, the glycoconjugate comprises less than about 20% free polysaccharides compared to the total amount of polysaccharides. In one embodiment, the glycoconjugate comprises less than about 15% free polysaccharides compared to the total amount of polysaccharides.

[0089] Multivalent polysaccharide-protein conjugate vaccines The multivalent pneumococcal immunogenic composition may comprise capsular polysaccharides from a Streptococcus pneumoniae serotype selected from at least one of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 conjugated to one or more carrier proteins, wherein the polysaccharides from at least one serotype are prepared using reductive amination in an aprotic solvent such as DMSO. The present invention contemplates multivalent pneumococcal immunogenic compositions having polysaccharides from at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 serotypes. Preferably, a saccharide from a particular serotype is not conjugated to multiple carrier proteins.

[0090] In certain embodiments, polysaccharides from at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 serotypes are prepared using reductive amination in an aprotic solvent such as DMSO.

[0091] In certain embodiments, one or more of serotypes 3, 6A, 6B, 7F, 18C, 19A, 19F or 23F are prepared using reductive amination in an aprotic solvent. In certain aspects of this embodiment, one or both of serotypes 3 or 18C are prepared using reductive amination in an aprotic solvent.

[0092] In certain embodiments, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the serotypes in the multivalent composition are formulated in an aprotic solvent, with the remaining serotypes being formulated using alternative chemistries and / or in aqueous solvents.

[0093] In certain embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, and 38 are prepared using reductive amination in an aprotic solvent. In certain embodiments, one or more of serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are prepared using reductive amination in an aprotic solvent. In certain embodiments, one or more of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35B, 35F and 38 are prepared using reductive amination in an aprotic solvent.

[0094] In one embodiment, the multivalent composition consists of polysaccharides from serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F prepared using reductive amination in an aprotic solvent such as DMSO, and polysaccharides from serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F prepared using reductive amination in an aqueous solvent.

[0095] After the individual glycoconjugates are purified, they are combined to form the immunogenic compositions of the invention. These pneumococcal conjugates are prepared by separate methods and combined in bulk into single dose formulations.

[0096] Carrier Proteins In certain embodiments of the present invention, the CRM 197 is used as a carrier protein. 197 is a non-toxic variant of diphtheria toxin (i.e., a toxoid). 197 is isolated from a culture of Corynebacterium diphtheria strain C7 (β197) grown in a casamino acid and yeast extract based medium. 197 is recombinantly prepared according to the methods described in U.S. Patent No. 5,614,382. 197 is purified by a combination of ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. 197 are prepared in Pseudomonas fluorescens using Pfenex Expression Technology™ (Pfenex, San Diego, Calif.).

[0097] Other suitable carrier proteins include further inactivated bacterial toxins such as DT (diphtheria toxoid) or fragment B of DT (DTFB), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g. as described in WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumococcal surface protein A (PspA; see WO 02 / 091998), pneumococcal adsorption protein (PsaA), C5a peptidase from group A or B streptococci, or Haemophilus influenzae protein D, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immunol. 2002, 1996, 1997, 1998). 63;2706-13) (ply detoxified in some way, e.g., dPLY-GMBS (see WO 04 / 081515) or dPLY-formol), PhtX (fusions of PhtA, PhtB, PhtD, PhtE and Pht proteins, e.g., PhtDE fusions, PhtBE fusions (see WO 01 / 98334 and WO 03 / 54007)) can also be used.Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB (from Neisseria meningitidis), PD (protein D of Haemophilus influenzae; see, for example, EP 0594610B) or immunologically functional equivalents thereof, synthetic peptides (see EP 0378881 and 0427347), heat shock proteins (see WO 93 / 17712 and WO 94 / 03208), pertussis proteins (see WO 98 / 58668 and EP 0471177), cytokines, lymphokines, growth factors or hormones (see WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al., 2001, Eur J Immunol 31:3816-3824), e.g., the N19 protein (see Baraldoi et al., 2004, Infect Immun 72:4884-7), iron uptake protein (see WO 01 / 72337), C. difficile toxin A or B (see WO 00 / 61761), and flagellin (see Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as carrier proteins.

[0098] Other DT variants, e.g., CRM 176 , CRM 228 , CRM 45 (Uchida et al.,1973,J Biol Chem 218:3838-3844);CRM 9 , CRM 45 , CRM 102 , CRM 103 and CRM 107and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletions or mutations of Glu-148 to Asp, Gln or Ser, and / or Ala158 to Gly, and other mutations disclosed in U.S. Patent Nos. 4,709,017 or 4,950,740; mutations of at least one or more of residues Lys516, Lys526, Phe530 and / or Lys534, and other mutations disclosed in U.S. Patent Nos. 5,917,017 or 6,455,673; or fragments disclosed in U.S. Patent No. 5,843,711 can be used as second carrier proteins. Such DT mutants can also be used to include epitope regions in DTFB mutants containing the B fragment.

[0099] When a multivalent vaccine is used, a second carrier can be used for one or more of the antigens in the multivalent vaccine. The second carrier protein is preferably a protein that is non-toxic and non-reactogenic and is available in sufficient quantity and purity. The second carrier protein is also conjugated or conjugated with the antigen, for example, Streptococcus pneumoniae polysaccharide, to enhance the immunogenicity of the antigen. The carrier protein should follow 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, each 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 typically conjugated to the same carrier protein.

[0100] Pharmaceutical / vaccine compositions The present invention further provides compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising, consisting essentially of, or consisting of any of the above combinations of polysaccharide serotypes, together with pharma- ceutically acceptable carriers and adjuvants.

[0101] Formulation of the polysaccharide-protein conjugates of the present invention can be achieved using art-recognized methods.For example, individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare a composition.Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

[0102] In one embodiment, the vaccine composition is formulated in an L-histidine buffer containing sodium chloride.

[0103] As defined herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of the immunogenic composition of the present invention. An immune adjuvant can enhance the immune response to an antigen that is weakly immunogenic when administered alone, for example, induce no or weak antibody titer or cellular immune response, increase the antibody titer to the antigen, and / or reduce the effective dose of the antigen to achieve an immune response in an individual. Thus, adjuvants are often administered to enhance the immune response and are well known to those skilled in the art. Adjuvants suitable for enhancing the effectiveness of a composition include, but are not limited to, the following:

[0104] (1) Aluminum salts (alum), for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate; (2) oil-in-water emulsion formulations (with or without muramyl peptides (defined below) or other specific immunostimulants such as bacterial cell wall components), such as (a) MF59 (WO 90 / 14837) containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (which may contain various amounts of MTP-PE) and formulated into submicron particles using a microfluidizer, such as a Model 110Y Microfluidizer (Microfluidics, Newton, Massachusetts); (b) 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L121, and SAF containing thr-MDP and either microfluidized to a submicron emulsion or vortexed to produce a larger particle size emulsion; (c) Ribi™ Adjuvant System (RAS) (Corixa, Hamilton, Mass.) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deacylated monophosphoryl lipid A (MPL™), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™), as described in U.S. Pat. No. 4,912,094; and (d) Montanide ISA; (3) Saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, Mass.) (see, e.g., U.S. Pat. No. 5,057,540) may be used, or particles produced from this adjuvant such as ISCOMs (immunostimulating complexes formed by the combination of cholesterol, saponin, phospholipids and amphipathic proteins) and Iscomatrix® (which have essentially the same structure as ISCOMs but do not contain the proteins); (4) bacterial lipopolysaccharide, synthetic lipid A analogs, such as aminoalkyl glucosamine phosphate compounds (AGPs), or derivatives or analogs thereof, available from Corixa and described in U.S. Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucopyranoside, also known as 529 (formerly known as RC529), formulated in aqueous form or as a stable emulsion; (5) synthetic polynucleotides, e.g., oligonucleotides containing CpG motif(s) (U.S. Pat. No. 6,207,646); (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18), 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; (7) Complement, for example, the trimer of complement component C3d.

[0105] In another embodiment, the adjuvant is a mixture of two, three or more of the above adjuvants, such as SBAS2 (an oil-in-water emulsion that also contains 3-deacylated monophosphoryl lipid A and QS21).

[0106] Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0107] 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), aluminum hydrate, aluminum trihydrate, hydrogel, Superfos, Amphogel, aluminum(III) hydroxide, aluminum hydroxyphosphate sulfate, aluminum phosphate adjuvant (APA), amorphous alumina, alumina trihydrate, or aluminum trihydroxy.

[0108] APA is an aqueous suspension of aluminum hydroxyphosphate. It is produced by blending aluminum chloride and sodium phosphate in a 1:1 volume ratio, precipitating aluminum hydroxyphosphate. After the blending process, the material is size reduced in a high shear mixer to achieve a monodisperse particle size distribution. The product is then diafiltered against saline and steam sterilized.

[0109] In certain embodiments, commercially available Al(OH) 3Proteins are adsorbed using aluminium hydroxide (e.g. Alhydrogel or Superfos, Denmark / Accurate Chemical and Scientific, Westbury, NY) at a ratio of 50-200 g protein / mg aluminum hydroxide. Protein adsorption, in another embodiment, depends on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with a low pI are more strongly adsorbed to positively charged aluminum ions than proteins with a high pI. Aluminum salts establish a reservoir of antigens that are slowly released over 2-3 weeks, may be involved in non-specific activation of macrophages and complement activation, and / or stimulate innate immune mechanisms (possibly via stimulation of uric acid). See, e.g., Lambrecht et al., 2009, Curr Opin Immunol 21:23.

[0110] Monovalent bulk aqueous conjugates are typically blended together and diluted to a target of 8 μg / mL for all serotypes except 6B, which is diluted to a target of 16 μg / mL. Once diluted, the batches are filter sterilized and an equal volume of aluminum phosphate adjuvant is aseptically added to a final target aluminum concentration of 250 μg / mL. The adjuvanted combined batches are filled into single-use 0.5 mL / dose vials.

[0111] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, such as a CpG-containing oligonucleotide, in particular a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN1826 available from Coley Pharmaceutical Group.

[0112] "CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide" and similar terms refer to a nucleotide molecule of 6-50 nucleotides in length that contains an unmethylated CpG moiety. See, e.g., Wang et al., 2003, Vaccine 21:4297. In alternative embodiments, any other art-accepted definition of the term is intended. CpG-containing oligonucleotides include modified oligonucleotides using any synthetic internucleoside linkages, modified bases and / or modified sugars.

[0113] Methods for the use of CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110.

[0114] Administration / Dosage The compositions and formulations of the invention can be used to protect or treat humans susceptible to infection, such as pneumococcal infection, by administering the vaccine by a systemic or mucosal route. In one embodiment, the invention provides a method of inducing an immune response against a S. pneumoniae capsular polysaccharide conjugate comprising administering to a human an immunologically effective amount of an immunogenic composition of the invention. In another embodiment, the invention provides a method of vaccinating a human against pneumococcal infection comprising administering to the human an immunologically effective amount of an immunogenic composition of the invention.

[0115] The optimal amount of components for a particular vaccine can be confirmed by standard testing involving observing appropriate immune responses in subjects.For example, in another embodiment, the dosage for human vaccination is determined by extrapolating animal testing to human data.In another embodiment, the dosage is determined empirically.The vaccine has been shown to be immunogenic in animal data of infant rhesus monkeys.

[0116] An "effective amount" of a composition of the invention refers to the dose required to induce antibodies that significantly reduce the likelihood or severity of infection with a microorganism, such as Streptococcus pneumoniae, during a subsequent challenge.

[0117] The methods of the invention can be used to prevent and / or alleviate the major clinical syndromes caused by microorganisms such as Streptococcus pneumoniae, including both invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

[0118] Administration of the compositions of the present invention may include one or more of injections by intramuscular, intraperitoneal, intradermal or subcutaneous routes; or mucosal administration to the oral / alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (as it can more effectively prevent nasopharyngeal carriage of pneumococcus, thereby reducing infection at the earliest stage).

[0119] The amount of conjugate in each vaccine dose is selected as an amount that induces an immune protective response without significant adverse effects. Such amount may vary depending on the serotype of pneumococcus. Generally, for polysaccharide-based conjugates, each dose contains 0.1-100 μg, particularly 0.1-10 μg, more particularly 1-5 μg of each polysaccharide. 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.

[0120] Optimal amounts of components for a particular vaccine can be ascertained by standard testing involving observation of appropriate immune responses in subjects. For example, in another embodiment, dosages for human vaccination are determined by extrapolating animal studies to human data. In another embodiment, the dosages are determined empirically.

[0121] In one embodiment, the dosage of 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 yet another embodiment, the dosage of alum salt is per μg of recombinant protein.

[0122] According to any of the methods of the invention, and in one embodiment, the subject is a human. In certain embodiments, the human subject is an infant (less than 1 year old), a toddler (about 12-24 months) or a child (about 2-5 years old). In other embodiments, the human subject is an elderly subject (e.g., over 50 years old or over 65 years old). The compositions of the invention are also suitable for use in older children, adolescents and adults (e.g., 18-45 years old or 18-65 years old).

[0123] In one embodiment of the method of the invention, the composition of the invention is administered as a single inoculation. In another embodiment, the vaccine is administered two, three, four or more times, sufficiently spaced apart. For example, the composition can be administered at 1, 2, 3, 4, 5 or 6 month intervals, or any combination thereof. The immunization schedule can be according to that specified for the pneumococcal vaccine. For example, the conventional schedule for infants and young children against invasive disease caused by Streptococcus pneumoniae is at 2, 4, 6 and 12-15 months of age. Thus, in one embodiment, the composition is administered as a four-dose series at 2, 4, 6 and 12-15 months of age.

[0124] Compositions of the invention may also include one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those identified in WO 02 / 083855 and WO 02 / 053761.

[0125] compound The compositions of the present invention may be administered to a subject by one or more methods known to those of skill in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally, etc., and may be formulated accordingly.

[0126] In one embodiment, the compositions of the invention are administered by epidermal, intramuscular, intravenous, intraarterial, subcutaneous, or intramucosal injection of a liquid formulation. Liquid formulations for injection include solutions and the like.

[0127] The compositions of the present invention may be formulated as single dose vials, multi-dose vials, or as pre-filled syringes.

[0128] In another embodiment, the composition of the present invention is administered orally, and is therefore formulated in a form suitable for oral administration, i.e., as a solid or liquid formulation. Solid oral formulations include tablets, capsules, pills, granules, pellets, etc. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, etc.

[0129] Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, such as saline and buffered media.Examples of oils are animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oils, or fats from milk or eggs.

[0130] Pharmaceutical compositions can be isotonic, hypotonic or hypertonic.However, it is often preferable that pharmaceutical compositions for infusion or injection are essentially isotonic when administered.Therefore, for storage, pharmaceutical compositions can be preferably isotonic or hypertonic.If pharmaceutical compositions are hypertonic for storage, they should be diluted to become isotonic before administration.

[0131] The isotonicity agent may be an ionic agent, such as a salt, or a non-ionic agent, such as a carbohydrate. Examples of ionic tonicity agents include NaCl, CaCl 2 , KCl and MgCl 2 Examples of non-ionic tonicity agents include, but are not limited to, mannitol, sorbitol, and glycerol.

[0132] It is also preferred that at least one pharma- ceutically acceptable additive is a buffer. For some purposes, for example when the pharmaceutical composition is for infusion or injection, it is often desirable for the composition to contain a buffer, which can treat the solution to a pH of 4 to 10, for example 5 to 9 (such as 6 to 8).

[0133] The buffer may, for example, be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffers.

[0134] Furthermore, the buffer may be selected from USP compatible buffers for parenteral use, for example, especially if the pharmaceutical formulation is for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids, such as acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid; dibasic acids, such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid; polybasic acids, such as citric acid and phosphoric acid; and bases, such as ammonia, diethanolamine, glycine, triethanolamine and TRIS.

[0135] Parenteral vehicles (for subcutaneous, intravenous, intraarterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of surfactants and other pharma-ceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, glycols 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 injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oils, or lipids from milk or eggs.

[0136] The formulations of the invention may also contain surfactants, such as polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), in particular PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX®; octoxynols, which vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, of which octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholines (lecithins); nonylphenol ethoxylates, such as the Tergitol® NP series; triethylene glycol monolauryl ether (Brij Polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as Span 30; and sorbitan esters, such as sorbitan trioleate (Span 85) and sorbitan monolaurate (commonly known as SPAN). A preferred surfactant for inclusion in the emulsion is PS-80.

[0137] A mixture of surfactants can be used, such as a PS-80 / Span 85 mixture. A combination of a polyoxyethylene sorbitan ester, such as polyoxyethylene sorbitan monooleate (PS-80), and an octoxynol, such as t-octylphenoxypolyethoxyethanol (Triton X-100), is also suitable. Another useful combination includes laureth 9 and a polyoxyethylene sorbitan ester and / or an octoxynol.

[0138] Preferred amounts (by weight) of surfactants are as follows: polyoxyethylene sorbitan esters (e.g. PS-80) 0.01-1%, in particular about 0.1%, octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001-0.1%, in particular 0.005-0.02%, polyoxyethylene ethers (e.g. Laureth 9) 0.1-20%, preferably 0.1-10%, in particular 0.1-1% or about 0.5%.

[0139] In certain embodiments, the composition consists essentially of histidine (20 mM), saline (150 mM), and 0.02% PS-20 or 0.04% PS-80 at pH 5.8, and 250 μg / mL APA (aluminum phosphate adjuvant). PS-20 can range from 0.005% to 0.1% (w / v) in the presence of PS-20 or PS-80 in a blend controlled aggregation during simulated manufacturing and shipping using primary packaging. The process consists of combining a blend of up to 24 serotypes in histidine, saline, and PS-20 or PS-80, then combining this blend material with APA and saline, with or without antimicrobial preservatives.

[0140] The choice of surfactant may need to be optimized for different drugs and drug substances. For multivalent vaccines with 15 or more serotypes, PS-20 and P188 are preferred. The choice of chemistry used to manufacture the conjugate may also play an important role in the stabilization of the formulation. In particular, when the conjugation reactions used to prepare the different polysaccharide-protein conjugates in the multivalent composition include both aqueous and DMSO solvents, we found that certain surfactant systems lead to significant differences in stability. Improved stability of polysorbate-protein conjugates was seen with polysorbate 20 alone or with poloxamer 188 in combination with a polyol.

[0141] The exact mechanisms by which specific detergents protect biological drugs are poorly understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential exclusion, competition for the air / liquid interface between the biological drug and the surface, surface tension, and / or direct association of the surfactant with the biological agent to hide hydrophobic patches that act as seeds for aggregation.

[0142] Poloxamers can also be used in the compositions of the present invention. Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by the trade name Pluronic®. Because the length of the polymer block can be customized, there are many different poloxamers with slightly different properties. For the general term "poloxamer", these copolymers are generally designated by the letter "P" (for poloxamer) followed by three digits, with the first two digits x 100 indicating the approximate molecular mass of the polyoxypropylene core. The last digit x 10 indicates the percentage of polyoxyethylene content (e.g., P407=poloxamer with a polyoxypropylene molecular mass of 4,000 g / mol and 70% polyoxyethylene content). Under the trade name Pluronic®, the coding of these copolymers begins with a letter (L=liquid, P=paste, F=flake (solid)) to define their physical form at room temperature, followed by two or three digits. The first digit of the numerical designation (two of three digits) multiplied by 300 indicates the approximate molecular weight of the hydrophobe, and the last digit x 10 indicates the percentage of polyoxyethylene content (e.g., L61=Pluronic® with a polyoxypropylene molecular mass of 1,800 g / mol and 10% polyoxyethylene content). See U.S. Pat. No. 3,740,421.

[0143] Exemplary poloxamers have the general formula: HO(C2 H 4 O) a (C 3 H 6 O) b (C 2 H 4 O) a H where the a and b blocks have the following values: Pluronic® Poloxamer ab Molecular Weight L31 2 16 1100 (average) L35 1900 (average) L44NF 124 12 20 2090~2360 L64 2900 (average) L81 2800 (average) L121 4400 (average) P123 20 70 5750 (average) F68NF 188 80 27 7680~9510 F87NF 237 64 37 6840~8830 F108NF 338 141 44 12700~17400 F127NF 407 101 56 9840~14600 As used herein, the molecular weight units are Daltons (Da) or g / mol.

[0144] Preferably, the poloxamer generally has a molecular weight in the range of 1100-17,400 Da, 7,500-15,000 Da, or 7,500-10,000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of the poloxamer in the formulation is 0.001%-5% weight / volume, or 0.025%-1% weight / volume. In certain embodiments, the polyol is propylene glycol and is at a final concentration of 1%-20% weight / volume. In certain embodiments, the polyol is polyethylene glycol 400 and is at a final concentration of 1%-20% weight / volume.

[0145] Suitable polyols for the formulations of the present invention are polymer polyols, particularly polyether diols, including but not limited to propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ether. Propylene glycol is available in a monomer molecular weight range of -425 to -2700. Polyethylene glycol and polyethylene glycol monomethyl ether are also available in a molecular weight range ranging from -200 to -35000, including but not limited to PEG 200, PEG 300, PEG 400, PEG 1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferred polyethylene glycol is polyethylene glycol 400. The final concentration of polyol in the formulations of the present invention may be 1% to 20% weight / volume or 6% to 20% weight / volume.

[0146] The formulation also contains a pH buffered saline. The buffer may be selected from the group consisting of, for example, TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffers. The buffer may treat the solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In some embodiments of the invention, the buffer is selected from the group consisting of phosphate, succinate, histidine, MES, MOPS, HEPES, acetate or citrate. Additionally, the buffer may be selected from, for example, USP compatible buffers for parenteral use, especially if the pharmaceutical formulation is for parenteral use. The concentration of the buffer may range from 1 mM to 50 mM or 5 mM to 50 mM. In certain embodiments, the buffer has a final concentration of 5 mM to 50 mM histidine or a final concentration of 1 mM to 10 mM succinate. In certain embodiments, the histidine has a final concentration of 20 mM ± 2 mM.

[0147] Although saline (i.e., a solution containing NaCl) is preferred, other salts suitable for the formulation include CaCl 2 , KCl and MgCl 2 , and combinations thereof. Non-ionic tonicity agents, including but not limited to sucrose, trehalose, mannitol, sorbitol and glycerol, can 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 embodiment, the saline is NaCl and may be present at a concentration of 20 mM to 170 mM.

[0148] In one embodiment, the formulation comprises an L-histidine buffer containing sodium chloride.

[0149] In certain embodiments of the formulations described herein, the polysaccharide-protein conjugate comprises one or more pneumococcal polysaccharides conjugated to a carrier protein. 197 , diphtheria toxin fragment B (DTFB), DTFBC8, diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, and combinations thereof. In one embodiment, all polysaccharide-protein conjugates are prepared using aqueous chemistry. In another embodiment, one or more polysaccharide-protein conjugates are prepared using DMSO solvent. As an example, the polysaccharide-protein conjugate formulation can be a 15-valent pneumococcal conjugate (15vPnC) formulation in which polysaccharide-protein conjugates from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are prepared using DMSO solvent, and polysaccharide-protein conjugates from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F are prepared using aqueous solvent.

[0150] In another embodiment, the pharmaceutical composition is delivered in a sustained release system. For example, the drug can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration. In another embodiment, polymeric materials are used, for example, in microspheres or implants.

[0151] Compositions of the invention may also include one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those identified in WO 02 / 083855 and WO 02 / 053761.

[0152] Although various embodiments of the present invention have been described with reference to the accompanying description and drawings, it should be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be made by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0153] The following examples illustrate, but do not limit, the present invention.

[0154] [Example] Example 1: Preparation of Streptococcus pneumoniae capsular polysaccharide Methods for culturing pneumococci are well known in the art. See, e.g., Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, e.g., European Patent No. 0497524. Isolates of pneumococcal subtypes are available from the American Type Culture Collection (Manassas, Virginia). The bacteria are identified as encapsulated, nonmotile, Gram-positive, lancet-shaped diplococci (alpha-hemolytic on blood agar). Subtypes can be differentiated based on the Quelling reaction using specific antisera. See, e.g., U.S. Patent No. 5,847,112.

[0155] A cell bank representing each S. pneumoniae serotype present was obtained in frozen vials from Merck Culture Collection (Rahway, NJ). The thawed seed culture was transferred to a seed fermentor containing pre-sterilized growth media appropriate for S. pneumoniae. The culture was grown in the seed fermentor with temperature and pH control. The entire volume of the seed fermentor was transferred to a production fermentor containing pre-sterilized growth media. The production fermentation was the final cell growth stage of the process. Temperature, pH and agitation speed were controlled.

[0156] The fermentation process was terminated by the addition of an inactivation agent. After inactivation, the batch was transferred to an inactivation tank and held under controlled temperature and agitation. A combination of centrifugation and filtration was used to remove cell debris. The batch was ultrafiltered and diafiltered. The batch was then subjected to solvent fractionation, which removed impurities and recovered the polysaccharides.

[0157] Example 2: CRM of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F using reductive amination in aqueous solution 197 Conjugation to Different polysaccharide serotypes were purified using a common process flow. 197 The polysaccharides are then solubilized, size-reduced, chemically activated, and buffer-exchanged by ultrafiltration. The purified CRMs are then conjugated to carrier proteins. 197 in the reaction mixture 2 (2 mM) was used to conjugate to the activated polysaccharide and the resulting conjugate was purified by ultrafiltration followed by final filtration through a 0.2 micron filter. Several process parameters within each step such as pH, temperature, concentration and time are controlled to serotype-specific values ​​in the following chapters.

[0158] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered through a 0.45 micron filter for all serotypes except serotype 19A. All serotypes except serotype 19A were homogenized to reduce the molecular mass of the polysaccharide. Serotype 19A was not size-reduced due to its relatively small starting size. To achieve serotype-specific molecular masses, the homogenization pressure and number of passes through the homogenizer were controlled to serotype-specific target values ​​(150-1000 bar; 4-7 passes). The size-reduced polysaccharide was filtered through a 0.2 micron filter, then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0159] The polysaccharide solution was then adjusted to a serotype-specific temperature (4-22 °C) and pH (4-5) with sodium acetate buffer to minimize size reduction of the polysaccharide due to activation. For all serotypes (except serotype 4), polysaccharide activation was initiated by the addition of 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype-specific and ranged from approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of the sodium metaperiodate was to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). For serotype 4, the batch was incubated at approximately 50 °C and pH 4.1 to partially deketalize the polysaccharide before adding sodium metaperiodate.

[0160] For all serotypes except serotypes 5 and 7F, the activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane. Serotypes 5 and 7F were diafiltered against 10 mM sodium acetate. Ultrafiltration for all serotypes was performed at 2-8 °C.

[0161] CRM 197 Conjugation of polysaccharides to The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate (pH 6.0 or pH 7.0), depending on the serotype. The pH of the selected buffer was to improve the stability of the activated polysaccharide during the conjugation reaction. Purified CRM obtained by expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876) was added to the 100-mL PBS. 197 Filter through a 0.2 micron filter and dilute with polysaccharide to CRM ranging from 0.4 to 1.0 w / w depending on the serotype. 197 The polysaccharide was combined with the buffered polysaccharide solution in a mass ratio of polysaccharide to CRM in the resulting conjugate. 197 Mass ratios were chosen to control the ratio. Polysaccharide and phosphate concentrations were serotype specific and ranged from 3.6 to 10.0 g / L and 100 to 150 mM, respectively, depending on the serotype. Serotype-specific polysaccharide concentrations were chosen to control the size of the resulting conjugates. The solutions were then filtered through a 0.2 micron filter. Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole of polysaccharide repeating unit) was added. Conjugation was allowed to proceed for a serotype-specific period (72 to 120 hours) to maximize polysaccharide and protein consumption.

[0162] Reduction with sodium borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g / L, cooled to 2-8 °C, and filtered through a 1.2 micron filter. All serotypes (except serotype 5) were diafiltered against 100 mM potassium phosphate (pH 7.0) using a 100 kDa NMWCO tangential flow ultrafiltration membrane at 2-8 °C. The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide / L and pH adjusted by the addition of 1.2 M sodium bicarbonate (pH 9.4). Sodium borohydride (1 mole per mole of polysaccharide repeating unit) was added. 1.5 M potassium phosphate (pH 6.0) was added later. Serotype 5 was diafiltered against 300 mM potassium phosphate using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

[0163] Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 using a 300 kDa NMWCO tangential flow ultrafiltration membrane at 4° C. The retentate batch was filtered through a 0.2 micron filter.

[0164] The serotype 19F conjugate was incubated at 22° C. for approximately 7 days, diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 100 kDa NMWCO tangential flow ultrafiltration membrane, and filtered through a 0.2 micron filter.

[0165] The batch was adjusted to a polysaccharide concentration of 1.0 g / L with an additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0. The batch was divided into aliquots and frozen at ≦−60° C.

[0166] Example 3: CRM of serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F using reductive amination in dimethyl sulfoxide 197 Conjugation Methods The different polysaccharide serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F were purified using the general process flow. 197 Conjugated individually to carrier proteins. Polysaccharides are dissolved, sized to the desired molecular mass, chemically activated, and buffer exchanged by ultrafiltration. Activated polysaccharides and purified CRM 197 were individually lyophilized and redissolved in dimethyl sulfoxide (DMSO). The redissolved polysaccharide solution and CRM were then 197 The solutions were combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration followed by a final filtration through a 0.2 micron filter. Several process parameters within each step, such as pH, temperature, concentration and time, are controlled to serotype-specific values ​​in the following chapters.

[0167] Size reduction and oxidation of polysaccharides Purified pneumococcal capsular Ps powder was dissolved in water and all serotypes except serotype 19A were filtered through a 0.45 micron filter. All serotypes except serotypes 18C and 19A were homogenized to reduce the molecular mass of Ps. Homogenization pressure and number of passes through the homogenizer were controlled to serotype-specific targets (150-1000 bar; 4-7 passes). Serotype 18C was size-reduced by acid hydrolysis at ≥90°C.

[0168] The size-reduced polysaccharide was filtered through a 0.2 micron filter, then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane. A 5 kDa NMWCO membrane was used for serotype 18C.

[0169] The polysaccharide solution was then adjusted to a serotype-specific temperature (4-22 °C) and pH (4-5) with sodium acetate buffer to minimize size reduction of the polysaccharide due to activation. For all serotypes (except serotype 4), polysaccharide activation was initiated by the addition of 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype-specific and ranged from approximately 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of the sodium metaperiodate was to achieve the target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). For serotype 4, the batch was incubated at approximately 50 °C and pH 4.1 to partially deketalize the polysaccharide before adding sodium metaperiodate.

[0170] The activated product was diafiltered against 10 mM potassium phosphate (pH 6.4) using a 10 kDa NMWCO tangential flow ultrafiltration membrane and then diafiltered or dialyzed against water using a 10 kDa NMWCO membrane. A 5 kDa NMWCO membrane was used for serotype 18C. Ultrafiltration or dialysis for all serotypes was performed at 2-8 °C.

[0171] CRM 197 Conjugation of polysaccharides to Purified CRM obtained through expression in Pseudomonas fluorescens as previously described (WO 2012 / 173876). 197 The solution was diafiltered against 2-5 mM phosphate (pH 7.0) buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and filtered through a 0.2 micron filter.

[0172] For serotypes other than serotype 3, the oxidized polysaccharide was formulated at 6 mg Ps / mL and 5% w / v sucrose in water (50 mg sucrose / mL). For serotype 3, the oxidized polysaccharide was formulated at 2 mg Ps / mL and 10% w / v sucrose in water (100 mg sucrose / mL). Protein solutions were formulated at 6 mg Pr / mL and 1% w / v sucrose in phosphate buffer (10 mg sucrose / mL).

[0173] Formulated Ps and CRM 197 The solutions were freeze-dried individually. Freeze-dried Ps and CRM 197 The materials were redissolved in DMSO and combined using a mixing tee. Sodium cyanoborohydride (1 mole per mole of polysaccharide repeating unit) was added and conjugation was allowed to proceed for a serotype-specific period (1-48 hours) to achieve the targeted conjugate size.

[0174] Reduction with sodium borohydride Sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added after the conjugation reaction. The batches were diluted in 150 mM sodium chloride with or without about 0.025% (w / v) polysorbate 20 at about 4° C. Potassium phosphate buffer was then added to neutralize the pH. For serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F and 33F, the batches were concentrated and diafiltered against 150 mM sodium chloride with or without 25 mM potassium phosphate (pH 7) at about 4° C. using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

[0175] Final Filtration and Product Storage Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 22F, 23F and 33F were concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with or without 0.015% (w / v) polysorbate 20 at 4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane. The retentate batch was filtered through a 0.2 micron filter.

[0176] Serotype 19F was incubated for approximately 5 days, diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0 at approximately 4° C. using a 300 kDa NMWCO tangential flow ultrafiltration membrane, and filtered through a 0.2 micron filter.

[0177] Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F and 33F were diluted with an additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, dispensed into aliquots and frozen at ≦−60° C.

[0178] Serotypes 4 and 14 were dialyzed against 150 mM sodium chloride using a 300 kDa NMWCO membrane at approximately 4°C, filtered through a 0.2 micron filter, dispensed into aliquots, and frozen at <-60°C.

[0179] Example 4: Analysis of the conjugates Molecular weight and concentration analysis of conjugates using HPSEC / UV / MALS / RI assay Conjugate samples were injected and separated by high performance size exclusion chromatography (HPSEC). Detection was achieved using ultraviolet (UV), multi-angle light scattering (MALS) and refractive index (RI) detectors in series. Protein concentrations were calculated from UV280 using the extinction coefficient. Polysaccharide concentrations were deconvoluted from the RI signal (contributed by both protein and polysaccharide) using the dn / dc coefficient, which is the change in refractive index of the solution and the change in solute concentration (reported in mL / g). The average molecular weight of the samples was calculated by Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using the measured concentrations and light scattering information across the entire sample peak.

[0180] Polysaccharide activation assay Conjugation occurs by reductive amination of activated aldehydes with lysine residues, mainly on carrier proteins. The level of activation, expressed as moles of aldehyde per mole of polysaccharide repeating unit, is important for controlling the conjugation reaction. An assay for measuring the degree of activation is described in US Patent Publication No. 2017 / 0021006.

[0181] An internal assay was developed to measure the degree of activation based on the reaction of aldehyde groups (generated during periodate oxidation of polysaccharides) with thiosemicarbazide (available from commercial sources).

[0182] Quantification can be achieved by NMR (nuclear magnetic resonance) or by comparing the derivatized polysaccharide to an appropriate reference standard and / or by using the extinction coefficient of the derivative. The use of extinction coefficients in this assay is similar to that used in the HPSEC / UV / MALS / RI method.

[0183] In general, the assay can be carried out under the following reaction conditions.

[0184] Time: 0.5 hours to 35 hours (this is serotype specific, but the reaction is allowed to continue to completion, i.e., until it plateaus in the time course) Temperature: 15-37°C, preferably around 21-27°C TSC concentration: 1~5mg / mL Reaction pH: pH 3 to 5.5, preferably 4.0 For Example 4, polysaccharides were derivatized with 1.25-2.5 mg / mL thiosemicarbazide (TSC) at pH 4.0 to introduce the chromophore (derivatization of activated polysaccharides for serotypes 1, 5, and 9V used 1.25 mg / mL TSC). The derivatization reaction was allowed to proceed until a plateau was reached. The actual time varied depending on the reaction rate of each serotype. TSC-Ps were then separated from TSC and other low molecular weight components by high performance size exclusion chromatography. Signal was detected by UV absorbance at 266 nm. Levels of activated aldehydes are calculated against a standard curve injection of Mono-TSC or directly using a given extinction coefficient. Mono-TSC is a synthetic thiosemicarbazone derivative of a monosaccharide. Aldehyde levels are then converted to moles of aldehyde per mole of repeating unit (Ald / RU) using Ps concentrations measured by HPSEC / UV / MALS / RI assay.

[0185] Similar derivatizations can be performed with thiosemicarbazide structural analogs, hydrazides, hydrazines, semicarbazides, semicarbazide structural analogs, aminooxy compounds, or aromatic amines, so long as the derivative has significant UV absorbance. The UV absorbance can be from a chromophore attached to the derivatizing agent, or a chromophore generated as a result of aldehyde derivatization, as in the case of thiosemicarbazide.

[0186] Determination of lysine consumption in conjugated proteins as a measure of the number of covalent bonds between polysaccharides and carrier proteins The extent of conjugation in the conjugate samples was measured using Waters AccQ-Tag Amino Acid Analysis (AAA). To degrade the carrier proteins into their component amino acids, samples were hydrolyzed using gas phase acid hydrolysis on an Eldex workstation. Free amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized samples were then analyzed using UPLC with UV detection on a C18 column. The average protein concentration was obtained using representative amino acids other than lysine. Lysine consumption during conjugation (i.e., lysine loss) was determined by the difference between the average measured lysine amount in the conjugate and the expected lysine amount in the starting protein.

[0187] Properties of conjugates prepared using reductive amination in aqueous and DMSO solutions Table 1 lists the results of polysaccharide activation and lysine consumption (i.e., lysine loss) for conjugates prepared using the processes described in Examples 2 and 3. There is a clear difference in that the conjugate prepared in DMSO (Example 3) has higher lysine consumption and lower polysaccharide activation than the conjugate prepared in aqueous solution (Example 2). This suggests that preparing the conjugate in DMSO solution allows the polysaccharide to be attached to more conjugation sites on the carrier protein with less activation or disruption to the native polysaccharide structure. As a result, due to higher cross-linking in the conjugate prepared in DMSO solution than in aqueous solution, the conjugate contains more glycopeptides per polysaccharide repeat unit on average. Glycopeptides are believed to be the antigenic domain against which the immune response is generated. As a result, the conjugates produced in DMSO are expected to be more immunogenic than those produced in aqueous solution.

[0188] The average molecular weight (Mw) of the conjugates in Table 1 was determined by HPSEC UV-MALS-RI assay. Conjugates produced by reductive amination in aqueous solution ranged from 990 to 3410 kDa. Conjugates produced in DMSO were generally larger, ranging in size from 1300 to 5822 kDa. [Table 1]

[0189] CRM 197 Quantification of the degree of conjugation at different sites on The polysaccharides are attached to the N-terminal amine groups of carrier proteins or CRMs. 197 The lysine residues can be conjugated to any of the side chains of the 39 lysine residues in the CRM. 197 The amino acid sequence of CRM is provided in Table 2, in which the lysine (abbreviated as K) is underlined and in bold. 197 To identify and quantify the extent of polysaccharide conjugation at different sites on the protein, an LC / UV / MS peptide mapping method was used. Representative conjugate samples (prepared in DMSO or aqueous solution) were double digested with trypsin to generate tryptic peptides. The mixtures were then subjected to reversed-phase C 18 The product was separated on a column and analyzed by UV and mass spectrometry. 197 Protein samples (not conjugated with polysaccharide) were also run in triplicate along with the controls. Trypsin cleaves proteins C-terminal to lysine and arginine residues, so conjugation at a lysine residue makes that site protease resistant. The extent of conjugation at a particular site can be determined by the CRM 197 It was determined by calculating the decrease in peak intensity of the tryptic peptide compared to the control. Depending on the cleavage site and sequence, the signal decrease of a particular peptide may be due to a miscleavage of a lysine in the preceding peptide, or a miscleavage of a lysine at the peptide terminus, or a conjugation in the middle of the peptide sequence.

[0190] CRM 197 The relative percentage of peptide signal reduction for serotype 19A conjugates compared to the control is plotted against the possible conjugation sites in Figure 1. Lysine positions listed on the x-axis represent the lysine positions of the CRM 197 The conjugation sites are numbered based on their order on the protein sequence and represent the possible conjugation sites of the analyzed peptides. For example, "33" means that the decrease in the peptide signal was due to conjugation at lysine 33, and "6,7" means that the decrease in the peptide signal was due to conjugation at lyines 6 and / or 7. The data in FIG. 1 suggested that not only was the degree of conjugation at each site generally higher for conjugates prepared in DMSO compared to aqueous solutions, but also that there were more conjugation sites in DMSO. These additional conjugation sites included lysines 29, 30, 31, and 32, the only lysines in previously identified common human T-cell peptide epitopes (see Raju et al., 1995, Eur. J. Immunol. 25:3207-3214; CRM 197 (Peptides 411-430 and 431-450 of the sequence). Similar results were observed with the other serotypes tested. [Table 2]

[0191] Example 5: Serotype 3Ps-CRM prepared in aqueous solution 197 Conjugates and serotype 3Ps-CRM prepared in DMSO 197 Mouse immunogenicity study comparing conjugates 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. Protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at MRL, West Point, PA.

[0192] Eight-week-old female CD1 mice were housed in microisolator cages (n=10 / cage) in the animal facility at MRL, West Point, PA. Food and water were available ad libitum. Mice (n=10 / group) were immunized with ST3-CRM formulated with aluminum phosphate adjuvant (APA) as described in Table 3. 197 Animals were immunized intramuscularly (IM) with the conjugate (0.4 μg of ST3 polysaccharide). Negative control animals received APA alone. Immunizations were performed on days 0, 14, and 28. On days 6 and 34, blood was collected via the tail vein into serum separator tubes (BD, Franklin Lakes, NJ). [Table 3]

[0193] Electrochemiluminescence (ECL) immunogenicity assay Mouse antibody responses were measured in a 96-well multiplex electrochemiluminescence assay as previously described with minor modifications. See Marchese et al., 2009, Clin Vaccine Immunol 16(3):387-96; Skinner et al., 2011, Vaccine 29(48):8870-6 and Caro-Aguilar et al., 2017 Vaccine 35(6):865-72. Briefly, test serum incubation was performed for 1 h on Meso-Scale Discovery plates (Meso Scale Diagnostics, Rockville, MD) and after washing, 25 μl of 2 μg / ml Sulfo-tag (Meso Scale Diagnostics, Rockville, MD)-labeled goat anti-mouse IgG was added to each well. Plates were incubated for 1 h at room temperature with shaking and then processed as previously described and read on a MESO Sector S600.

[0194] ECL titers were calculated as the reciprocal of the linearly interpolated dilution corresponding to the cutoff value (pneumococcal polysaccharide ECL geometric mean signal of a given positive control pooled mouse serum). Interpolation was performed using the logarithmic scale of ECL and dilution. Titers were then obtained by back-transforming the linearly interpolated dilution. Titers were extrapolated for samples falling outside the test dilution range of 100 to 1,562,500 based on a linear extrapolation (log-log scale) using the intercept and slope of the last three ECL data points for sample curves completely above the cutoff line, or the intercept and slope of the first two ECL data points for sample curves completely below the cutoff line. Titers were then obtained by back-transforming the linearly extrapolated dilution.

[0195] Opsonophagocytic killing assay (OPA) Opsonophagocytic killing assays (OPA) of pneumococcus serotype 3 were performed as previously described with minor modifications (Caro-Aguilar et al., 2017 Vaccine 35(6):865-72 and Burton et al., 2006, Clin Vaccine Immunol 13(9):1004-9). After incubation of serum, bacteria, complement and HL-60 cells, 10 μl of the opsonophagocytic reaction was transferred to individual wells on a Millipore 96-well filter plate containing 200 μl / well of sterile water. The plate was vacuum filtered and 100 μl of Todd Hewett yeast extract (THYE, Teknova) broth was added. The media was filtered and the wet plate was placed in a sealed plastic bag overnight at 27 °C. Plate filters were then stained with 100 μl / well of 0.1% Coomassie Blue solution (Bio-Rad, Hercules, CA). The stain was filtered through the plate, and colonies were destained with Coomassie decolorizing solution (Bio-Rad) and vacuum filtered again to dryness. Stained bacterial colonies were counted in a CTL Immunospot reader (Shaker Heights, OH). OPK titers were defined as the reciprocal of the serum dilution with at least 50% killing compared to the average growth in complement control (no serum control) wells and were calculated by linearly interpolating between the serial dilutions whose signal bracketed 50% killing.

[0196] The results before and after the third dose are shown in Figure 2 and Table 4 for ECL immunogenicity and in Figure 3 for OPA. Both conjugates prepared by the process using aqueous and DMSO solutions are immunogenic and provide functional killing activity against bacteria. Interestingly, the conjugate prepared by the process using DMSO solution had both higher ECL immunogenicity and OPA response than the conjugate prepared using aqueous solution. The difference in ECL immunogenicity is statistically significant. The GMT ratio of group 3 to group 2 is 3.41 (lower and upper limits of 95% confidence interval are 1.26 and 9.26, respectively). [Table 4]

[0197] Example 6: Immunogenicity study in adults comparing pneumococcal polysaccharide-protein conjugates prepared using reductive amination in aqueous solution with those prepared using reductive amination in DMSO This example describes the immunogenicity and safety of two 15-valent pneumococcal conjugate vaccines (PCV15) in healthy pneumococcal vaccine-naive adults aged 50 years or older.

[0198] Test Design In healthy adult subjects aged 50 years or older (who were required to have documented stable underlying chronic disease), two different PCV15 formulations (PCV15-A and PCV15-B) and Prevnar 13™ (pneumococcal 13-valent conjugate vaccine [diphtheria CRM 197 A randomized, multicenter, double-blind study was conducted in accordance with Good Clinical Practices to compare the safety, tolerability, and immunogenicity of a single dose of 100 mg ...

[0199] A total of 690 healthy, pneumococcal vaccine-naive individuals aged 50 years or older were enrolled and randomized in a 1:1:1 ratio to three different vaccination arms: Prevnar 13™, PCV15-A, and PCV15-B. Randomization was stratified by age at study entry (50-64 years, 65-74 years, and ≥75 years).

[0200] PCV15 is CRM 197 Pneumococcal polysaccharides of each of the following serotypes (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F) at a dose of 2 μg / 0.5 mL conjugated to CRM 197The PCV15-A and PCV15-B formulations contained 4 μg / 0.5 mL dose of serotype 6B pneumococcal polysaccharide conjugated to 100 μg / 0.5 mL of 100 μg / 0.5 mL of aluminum phosphate adjuvant, 20 mM L-histidine, and 150 mM sodium chloride (pH 5.8). PCV15-A was formulated with 0.2% w / v P188. PCV15-B was formulated with 0.1% w / v PS-20.

[0201] For PCV15-A, all 15 polysaccharide serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F) were synthesized using reductive amination in aqueous solution as described in Example 2. 197 The attributes for some of these conjugates (Conjugate Lot No. 1 material) are listed in Table 1.

[0202] PCV15-B, serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F were purified using reductive amination in DMSO as described in Example 3. 197 The attributes of these conjugates (Conjugate Lot No. 2 material) are listed in Table 1. The conjugates for the remaining serotypes (1, 3, 4, 5, 9V, 14, 22F and 33F) are the same conjugates used for PCV15-A.

[0203] Both PCV15 formulations had a generally comparable safety profile to Prevnar 13™ based on cumulative safety assessments (data not shown). Serotype-specific IgG GMCs and OPA GMTs were measured at day 30. (OPA results not included).

[0204] result IgG geometric mean concentrations (GMC) and confidence intervals (CI) are summarized in Table 6. Serotype 6A, 6B, 7F, 18C, 19A, 19F and 23F conjugates in PCV15-A and PCV15-B were produced using different conjugation processes as described above. Consistent with the results shown in Table 4, the immunogenic response for each serotype shown in Table 6 was significantly greater when the polysaccharide serotype was treated with CRMP in DMSO. 197 The GMCs of serotypes 18C, 19A, 19F and 23F in PCV15-B were significantly higher than those in PCV15-A (two-sided α=0.05). These data strongly demonstrate the benefit of conjugation in DMSO to improve immunogenicity. This finding has not been previously demonstrated for pneumococcal or other conjugate vaccines. [Table 5]

Claims

[Claim 1] 1. A method for producing an immunogenic composition comprising a polysaccharide from Streptococcus pneumoniae serotype 3 conjugated to a carrier protein, wherein a conjugation reaction for conjugating the polysaccharide to the carrier protein is carried out in an aprotic solvent, the carrier protein being CRM 197 , the conjugation reaction being a reductive amination conjugation reaction, and the aprotic solvent being dimethylsulfoxide (DMSO).

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