Enhanced immunogenicity of Streptococcus pneumoniae polysaccharide-protein conjugates

Abstract

To provide a method for preparing a polysaccharide-protein conjugate.SOLUTION: A method for preparing a pneumococcal serotype 3 polysaccharide-CRM197 carrier protein conjugate by reductive amination includes: a) allowing pneumococcal serotype 3 polysaccharide to react with oxidizer to form activated pneumococcal serotype 3 polysaccharide; and b) allowing the activated pneumococcal serotype 3 polysaccharide to react with CRM197 carrier protein in aprotic solvent to form a pneumococcal serotype 3 polysaccharide-CRM197 carrier protein conjugate. The obtained conjugate has higher antigenicity measured by an opsonin phagocytic extinction assay and an electrochemiluminescence immunogenic assay compared to the pneumococcal serotype 3 polysaccharide-CRM197 carrier protein conjugate prepared according to the processes a) and b) in which the activated pneumococcal serotype 3 polysaccharide is allowed to react with CRM197 carrier protein in aqueous solution.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a reductive aryl group in an aprotic solvent such as dimethyl sulfoxide (DMSO). CRM using minification 197 at least one Streptococcus pneumoniae (S and (iii) providing an immunogenic composition comprising a streptococcus pneumoniae polysaccharide. The present invention also relates to a method for treating a pneumococcus Streptococcus aureus (S. pneumoniae) capsule comprising administering to a patient a therapeutically effective amount of one or more polysaccharides derived from the S. pneumoniae capsule in the presence of a non-promoter such as DMSO. Conjugated to carrier proteins using reductive amination performed in an aqueous solvent Enhancing the immunogenicity of immunogenic compositions having one or more polysaccharide-protein conjugates This provides a method for [Background technology]

[0002] Streptococcus pneumoniae is a gram-positive bacterium that is responsible for invasive bacterial diseases in infants and children (pneumonia, bacteremia, meningitis, Pneumococci are the most common cause of serotype specificity. There are over 90 known serotypes of Streptococcus pneumoniae, each of which is encapsulated by a polysaccharide that is specifically linked to the vesicle. The membrane not only protects the inner surface of the bacterium from complement but is also poorly immunogenic in itself, making it a key factor in preventing pneumonia. Polysaccharides are T-cell-independent antigens, so they interact with T cells. However, they cannot be processed or presented on MHC molecules in a manner that allows them to interact with one another. They may stimulate the immune system through other mechanisms involving cross-linking of surface receptors on B cells. This can be done.

[0003] Children under 2 years of age do not mount an immune response to most polysaccharide vaccines, so It is necessary to render polysaccharides immunogenic by chemical conjugation to protein carriers. Coupling polysaccharides (T-cell independent antigens) to proteins (T-cell dependent antigens) This allows for the development of T cell-dependent properties including isotype switching, affinity maturation, and memory induction. is given to the polysaccharide.

[0004] Many conjugation reactions are used to covalently attach polysaccharides to proteins. The three more commonly used methods include: 1) reductive Amination: An aldehyde or ketone group on one component of a reaction converts to an amino or ketone group on the other component. reacts with the hydrazide group, and the resulting C=N double bond is subsequently converted to a C=N single bond by a reducing agent. 2) Cyanylation conjugation: When polysaccharides are conjugated with cyanogen bromide (CNBr) or or 1-cyano-4-dimethylammonium pyridinium tetrafluoroborate (CD The cyanate group is introduced into the hydroxyl group by activation with either hydroxyl group or hydroxyl group. Upon addition of a protein component, it forms a covalent bond with the amino or hydrazide group. ) Carbodiimide reaction: A carbodiimide reacts with a carboxyl group on one of the components of the conjugation reaction. The activated carboxyl group is then bonded to the amino or hydra group on the other component. These reactions also react with the dihydrogen group of the conjugate prior to the conjugation reaction. It is also often used to activate the components of

[0005] Reductive amination has been utilized to conjugate Streptococcus pneumoniae polysaccharides. For example, U.S. Patent No. 8,192,746 and U.S. Patent Application Publication No. 20170021006 No. 2011 / 110381 and WO 2015 / 110941 See, Reductive amination involves two steps: (1) oxidation of the antigen, and (2) oxidation of the antigen. 2) Formation of a conjugate by reduction of the antigen and carrier protein. The reduction step is carried out in an aqueous solvent. Alternatively, the reaction may be carried out in an aprotic solvent such as DMSO. See issue / 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 Streptococcus pneumoniae serotypes 1, 2, 3, and 4 conjugated to a carrier protein. , 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15 C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 2 Polysaccharides derived from one or more of 8A, 31, 34, 35A, 35B, 35F and 38 an immunogenic composition comprising a conjugate in which the polysaccharide is conjugated to the carrier protein; The immunogenic composition is provided wherein the jugation reaction is carried out in an aprotic solvent. In an embodiment, for compositions having the same serotype, the compositions are prepared in an aprotic solvent. One or more serotypes prepared under aqueous conditions were compared to the same one or more serotypes prepared under aqueous conditions. Highly immunogenic.

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

[0009] The present invention also relates to 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, and conjugating the polysaccharide derived therefrom to a carrier protein. and conjugating the polysaccharide to the carrier protein in an aprotic solvent. A method is provided.

[0010] The present invention also provides a method for the preparation of Streptococcus pneumoniae serotypes 1, 2, and 3 conjugated to a carrier protein. , 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 1. A method of treating a subject with an immunogenic composition comprising: Also provided is a method in which the antibody is conjugated to the carrier protein in

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

[0012] In certain embodiments, derived from one or more of Streptococcus pneumoniae serotypes 3 and 18C The polysaccharide is conjugated to a carrier protein in an aprotic solvent. In one embodiment, the polysaccharide from Streptococcus pneumoniae serotype 3 is In one aspect of this embodiment, the pneumonia-associated The polysaccharide from Streptococcus serotype 18C was conjugated to a carrier protein in aprotic solvents. Gated.

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

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

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

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

[0017] In certain embodiments, the immunogenic composition is a polypeptide conjugated to a carrier protein. Streptococcus pneumoniae serotypes 6A, 6B, 7F, 10A, 15B, 19A, 19F, 22F, 23 and a polysaccharide derived from one or more of 33F and 33F, wherein the polysaccharide is attached to a carrier protein. The conjugation reaction is carried out in an aprotic solvent. In certain aspects of the embodiment, the conjugation reaction is reductive amination. In certain embodiments, the aprotic solvent is DMSO. Quality is CRM 197 In one embodiment, the immunogenic composition comprises a carrier protein. Conjugated Streptococcus pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and and 23F, and Conjugating a polysaccharide derived from 9A, 19F or 23F to the carrier protein The conjugation reaction is carried out in an aprotic solvent. In certain embodiments, the polysaccharide is It is derived from Streptococcus pneumoniae serotype 18C, 19A, 19F or 23F.

[0018] In certain embodiments, the immunogenic compositions of the invention are conjugated to a carrier protein. Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, and 7C , 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 1 6F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B , 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38, and the 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, 2 0, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 3 Polysaccharides derived from 3F, 34, 35A, 35B, 35F or 38 are coupled to the carrier protein. The conjugation reaction is carried out in an aqueous solvent. In the immunogenic composition, 35-100% of the serotypes were reduced amino acids under DMSO conditions. The remaining polysaccharide-protein conjugates were prepared under aqueous conditions. will be done.

[0019] In one particular embodiment, the present invention provides a CRM 197 Polysaccharide-conjugated lung Streptococcus pyogenes serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 1 An immunogenic composition consisting essentially of polysaccharides derived from 9F, 22F, 23F and 33F. Therefore, the Streptococcus pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F The conjugation reaction was carried out under DMSO conditions, and the Streptococcus pneumoniae serotypes 1 and 3 The conjugation reactions of 4, 5, 9V, 14, 22F, and 33F were carried out in aqueous media. and providing an immunogenic composition which may further comprise about 0.2% w / v PS-20. .

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

[0021] The present invention also provides a method for enhancing the efficacy of pneumococcal polysaccharide (PnP) protein conjugate vaccines. A method for providing an enhanced immune response, comprising conjugating a medicament to one or more carrier proteins. Streptococcus pneumoniae capsular polyclonal antibodies derived from a first set of two or more gated Streptococcus pneumoniae serotypes administering to an animal subject an immunogenic composition comprising a polysaccharide-protein conjugate comprising a sugar; wherein two or more of the polysaccharide-protein conjugates from the first set are In one embodiment, the compound is prepared by reductive amination under DMSO conditions. wherein the enhanced immune response is induced by two or more polysaccharide-protein antibodies derived from a first set. One or more of the conjugates is prepared using reductive amination under aqueous conditions. In one embodiment, the control animal is given the immunogenic composition. In another embodiment, the control animal is a human. The method further comprises administering a second antibody of a pneumococcal serotype conjugated to one or more carrier proteins. Further polysaccharide-protein conjugates containing Streptococcus pneumoniae capsular polysaccharides derived from the set are The present invention uses a pneumococcal polysaccharide protein conjugate vaccine comprising the polysaccharide-protein conjugate vaccine. Protein conjugates were prepared using reductive amination under aqueous conditions, and a second set of The serotypes derived from are different from the first set of serotypes.

[0022] In certain embodiments, the method comprises the step of: A, 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, 3 Pneumococcal polysaccharide protein conjugates selected from 5A, 35B, 35F and 38 Use a vaccine.

[0023] In certain embodiments, the method comprises the step of preparing a polysaccharide-protein derived from serotype 3 or 18C. Pneumococcal polysaccharide conjugates are prepared using reductive amination under DMSO conditions A protein conjugate vaccine is used.

[0024] In certain embodiments, the method comprises the step of: Protein conjugates are available for serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23 F.

[0025] In certain embodiments, the method comprises the step of: Serum protein conjugates were prepared using reductive amination under DMSO conditions. A second set of serotypes includes 6A, 6B, 7F, 18C, 19A, 19F, and 23F. The polysaccharide-protein conjugates derived from pneumococcal polysaccharides are prepared under aqueous conditions. Protein conjugate vaccines are used.

[0026] In one particular embodiment, the method comprises detecting serotypes 6A, 6B, 7F, 18C, 19A, Polysaccharide-protein conjugates derived from 19F and 23F were reduced under DMSO conditions. Prepared using selective amination, it is available for serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33 Polysaccharide-protein conjugates derived from pneumococcal polysaccharide protein prepared under aqueous conditions Use a protein conjugate vaccine.

[0027] In certain embodiments, the method comprises the step of obtaining polysaccharide proteins from 35 to 100% of the serotypes. The hydroxyl group conjugate was prepared using reductive amination under DMSO conditions, and the remaining Pneumococcal polysaccharide-protein conjugates prepared under aqueous conditions In one embodiment, a polyclonal vaccine derived from 45 to 80% of the serotypes is used. Glycoprotein conjugates were prepared using reductive amination under DMSO conditions, and and the remaining polysaccharide-protein conjugate is prepared under aqueous conditions. Polysaccharide-protein conjugates derived from 75–100% of serotypes were synthesized under DMSO conditions. and the remaining polysaccharide-protein conjugate is prepared using reductive amination of It is prepared under conditions.

[0028] In certain embodiments, the method comprises administering to a mammalian animal the carrier protein comprising Neisseria meningitidis. meningitides outer membrane protein complex (OMPC), tetanus toxoid, Diphtheria toxoid, protein D and CRM 197Pneumonia selected from the group consisting of A coccal polysaccharide protein conjugate vaccine is used. In one embodiment, the carrier protein Pak quality is CRM 197 is.

[0029] In certain embodiments, the method involves preparing hydroxybenzoates using reductive amination under DMSO conditions. The conjugate is determined by a protein lysine loss value greater than 5.0. In the case of pneumococcal polysaccharide proteins with a higher proportion of formed glycopeptide bonds, In one aspect of this embodiment, a conjugate vaccine is used. Conjugates prepared using reductive amination at 7.0–18, inclusive, were In another embodiment, reductive amination under DMSO conditions is used to obtain a lysine loss value. The conjugates prepared were 9.0, 9.1, 9.2, 9.3, 9.4, 9.5 or have a lysine loss value greater than 10.0.

[0030] In another specific embodiment, the present invention provides a CRM 197 Polysaccharide-conjugated pneumonia Coccus serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, Pneumococcal polysaccharide (PnP) protein consists essentially of polysaccharides derived from 22F, 23F, and 33F. A method for providing an enhanced immune response to a protein conjugate vaccine. Thus, polysaccharide-protein complexes derived from the first and second sets of pneumococcal serotypes are administering to a human subject an immunogenic composition comprising an adjuvant, The compound consists of 6A, 6B, 7F, 18C, 19A, 19F and 23F, and DMS A second set of serotypes, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, , 9V, 14, 22F and 33F, and prepared under aqueous conditions. To provide.

[0031] In certain embodiments, the immunogenic compositions produced by the methods of the present invention are used in vaccines. Enhanced immune responses in inoculated animals were observed in serum IgG or opsonophagocytic antibodies. In one embodiment, the increase in pneumococcal serotypes is measured by the geometric mean titer of the antibody. The enhanced immune response was observed in the polysaccharide-protein complex derived 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 human subjects 50 years of age or older. In certain embodiments, the method is used on human subjects aged 2 years or younger. In an embodiment, the method is used on an immunocompromised human subject.

[0033] The present invention also provides a method for preparing pneumococcal polysaccharide-protein conjugates by reductive amination. A method of a) Serotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 2 Streptococcus pneumoniae polysaccharides selected from 3F and 33F are treated with an amount of an oxidizing agent (e.g., periodate). salt) to form an activated polysaccharide having an activation level of 0.05 to 0.22; b) coupling the activated polysaccharide with a carrier protein in an aprotic solvent, optionally in the presence of a reducing agent; reacting the polysaccharide with a protein to form a polysaccharide-protein conjugate; The conjugate has a lysine loss value within the range of 7.0 to 18.0, inclusive. Provide the law.

[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 determined by the addition of thiosemicarbazide to the alkyl groups on the polysaccharide. It is measured by derivatizing the aldehyde.

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

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

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

[0040] Derivatizing agents include thiosemicarbazide, thiosemicarbazide structural analogs, hydrazides, and hydrazides. hydrazine, semicarbazide, semicarbazide structural analogues, aminooxy compounds or aromatic compounds The amine may be selected from the group consisting of aromatic amines.

[0041] In one embodiment, the quantification in step c) is by comparison to a derivative standard. In some embodiments, the quantification in step c) is by measurement relative to a predetermined extinction coefficient. do. [Brief explanation of the drawings]

[0042] [Figure 1] The extent of conjugation at different lysine sites on CRM197 was 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. The loss of peptide signal compared to the CRM197 control sample was plotted against the conjugation site. [Figure 2] Electrochemiluminescence (ECL) immunogenicity results from mouse study groups comparing serotype 3Ps-CRM197 conjugates prepared by reductive amination in aqueous solution or DMSO. The conjugates were 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. [Figure 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 INVENTION

[0043] The present invention relates to a method for treating a pneumococcal conjugate comprising administering to a subject a subject a conjugate derived from at least one serotype of pneumococcus, such as DMSO. Pneumococcal polysaccharide-protein complexes prepared using reductive amination in aprotic solvents The present invention provides immunogenic compositions comprising the polysaccharide-protein conjugates. DMSO, used as the solvent during the reductive amination of the gates, was prepared under aqueous conditions. The unexpectedly superior stability and The present invention is based in part on the discovery that carrier proteins confer enhanced immunogenicity. Enhanced covalent association of polysaccharides to proteins by direct consumption of lysine residues on the surface of Regarding the advantages of DMSO solvent above, for most serotypes tested, Conjugation in aprotic solvents results in lower polysaccharide activation levels than in The "lysine loss" that gives good immunogenicity (≧7.0) was achieved with a lysine loss ratio of 0.05 to 0.22. The increased covalent association increased the stability of the polysaccharide-protein conjugate. and enhance the immune response to those specific polysaccharide antigens conjugated in DMSO. have a direct interest in

[0044] Without being bound by any theory, the immunological effects observed with glycoconjugates prepared in DMSO One possible mechanism for enhanced immunogenicity is the use of ricin on the surface of the carrier protein. As the number of bonds between the residues and the carbohydrate (capsular polysaccharide) increases, the number of binding points between the protein and the polysaccharide increases. , thereby conferring stability and preventing the disruption or chemical decomposition of peptide carbohydrate bonds. For example, Hsieh, Characterization n of Saccharide-CRM 197 Conjugate Vaccine s in Brown F,Corbel M,Griffiths E(eds):P hysico-Chemical Procedures for the Chara cterization of Vaccines.Dev.Biol.Basel,K See Arger, 2000, Vol. 103, pp. 93-104. Further, the increased number of polysaccharide-protein bonds created during conjugation in the medium The advantage may be an increased likelihood of successful presentation of peptide-carbohydrate to T cells. Genetic variability in the human population is related to specific peptides conjugated to carbohydrate antigens. The association with or loading of the sequence results in variations in sensitivity or capacity. This increases the number of binding sites on the carrier protein, leading to successful antigen presentation on the APC surface. Increased likelihood of T-cell-dependent responses to other T-cell-independent antigens Enhanced immunogenicity observed by conjugation in DMSO solvent Another possible mechanism for the formation of CRM in organic solvents is 197 This is due to degeneration. This modification may make additional lysines available for polysaccharide binding, increasing glycosylation on the APC surface. This increases the likelihood of peptide presentation and T cell-dependent responses to different peptide epitopes. Avci et al., 2011, Nature Medicine 17 :1602-1610.

[0045] Denature CRM during conjugation 197 Conjugation in organic solvents to produce Yet another benefit is that it contains natural CRM 197 Reduced immunological interference of antibodies against epitopes The increased polysaccharide-protein bond produced during conjugation in DMSO solvent An additional advantage of this method is that larger sized polysaccharide-protein conjugates are formed, The compositions of the present invention may be useful in eliciting a human response, resulting in enhanced immunogenicity. It is believed to offer significant advantages.

[0046] As shown in Example 5, reductive amination in DMSO was used to produce Streptococcus pneumoniae serotype 3 Polysaccharide-protein conjugates prepared from (prepared using reductive amination in water) Mouse phagocytic activity (OPA) measured by phagocytic activity (compared to the same conjugate prepared in vitro) Furthermore, as shown in Example 6, the reduction in DMSO showed increased immunogenicity in a mouse model. Seven serotypes were prepared using reactive amination (and eight others were prepared in aqueous media). The 15-valent pneumococcal conjugate vaccine, which contains 15 serotypes, The 15-valent PCV prepared in DMSO was significantly higher than the corresponding 15-valent PCV prepared in a neutral solvent. For all seven serotypes, the results in humans (with four serotypes showing statistical significance) There was a tendency for the antibody to show superior immunogenicity in

[0047] As shown in Example 4, the serotype 19A conjugates (compared to the CRM197 control) CRM in progress 197 The peptide signal reduction for lysine positions on the protein can be Previously identified common conjugation sites were plotted against available conjugation sites. A novel human T cell peptide epitope reveals additional conjugation sites (Raju et al., 1995, Eur. J. Immunol. 25:32 See 07-3214; CRM 197Peptides 411-430 and peptide 43 of the array 1-450). Thus, in certain embodiments, the invention also provides one or more polysaccharide-CRM 197 conjugate-containing immunogenic composition, wherein the polysaccharide-CRM 19 At least one of the 7 conjugates is prepared in an aprotic solvent, and the conjugate prepared in the aprotic solvent exhibits greater accessibility of lysine residues in amino acids 411-430 or 431-450 of CRM compared to the same conjugate prepared in an aqueous solvent. The invention also relates to an immunogenic 197 composition comprising one or more polysaccharide-CRM conjugates, wherein at least one of the polysaccharide-CRM conjugates is prepared in an aprotic solvent, and one or more lysine residues in amino acids 411-430 or 431-450 197 of CRM in the conjugate prepared in the aprotic solvent are conjugated at more than 10%. In certain embodiments, the invention relates to a method for enhancing the accessibility of lysine residues in amino acids 197 411-430 or 431-450 of CRM by conjugating a polysaccharide to CRM in an aprotic solvent. In a particular aspect of this embodiment, one or more lysine residues in amino acids 411-430 or 431-450 of CRM in the conjugate prepared in the aprotic solvent are conjugated at more than 10%. 197 In certain embodiments, the invention relates to a method for enhancing the accessibility of one or more lysine residues in amino acids 411-430 or 431-450 of CRM, which comprises conjugating a polysaccharide to CRM in an aprotic solvent. In a particular aspect of this embodiment, one or more lysine residues in amino acids 411-430 197 or 431-450 of CRM in the conjugate prepared in the aprotic solvent are conjugated at more than 10%. The invention also relates to a method for enhancing the accessibility of lysine residues in amino acids 197 411-430 or 431-450 of CRM, especially within CRM 197 by conjugating a polysaccharide to CRM in an aprotic solvent. In a particular aspect of this embodiment, one or more lysine residues in amino acids 411-430 or 431-450 of CRM in the conjugate prepared in the aprotic solvent are conjugated at more than 10%. 197 In a particular aspect of this embodiment, one or more lysine residues in amino acids 411-430 or 431-450 of CRM in the conjugate prepared in the aprotic solvent are conjugated at more than 10%. In these embodiments, the polysaccharide is conjugated to a saccharide selected from the group consisting of saccharides, saccharides, and saccharides used in preparing the immunogenic composition. In certain embodiments, the polysaccharide may be derived from any organism suitable for inhibiting meningitis. The polysaccharides may be derived from any serotype of these organisms. .

[0048] As used herein, the term "aqueous solvent" or "aqueous conditions" refers to a process such as reductive amination. If used with any conjugation, solvent for the conjugation reaction This refers to the use of water as a buffer and a sol- uid, except that no organic solvents are present. Other ingredients may also be included.

[0049] As used herein, the term "aprotic solvent" refers to a solvent that is suitable for reactions such as reductive amination. When used with conjugation, as a solvent for the conjugation reaction, Refers to the use of polar aprotic solvents or combinations of polar aprotic solvents. Examples of protic solvents include dimethyl sulfoxide (DMSO), dimethylformamide ( Examples include, but are not limited to, hexamethylphosphoramide (DMF) and hexamethylphosphoramide (HMPA). Aprotic solvents may contain, for example, up to 1%, 2%, 5%, 10% or 20% Some water may be present.

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

[0051] As used herein, the term "comprises" refers to the immunogenicity of the present invention. When used with respect to the parenteral composition, the inclusion of any other ingredients such as adjuvants and excipients (Subject to the restriction of the phrase "consisting of" for antigen mixtures) The term "consisting of" refers to a polyvalent polysaccharide-protein When used with respect to a mixture of protein conjugates, the mixture is Other pneumococcal polysaccharides with glycoprotein conjugates but from different serotypes It refers to the absence of protein conjugates.

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

[0053] As used herein, the term "polysaccharide" is used interchangeably in the fields of immunological and bacterial vaccines. The term "antigenic sugar" is intended to encompass any commonly used antigenic sugar element (or antigenic unit). "sugar", "oligosaccharide", "polysaccharide", "liposaccharide", "lipo-oligosaccharide (LOS)", "lipopolysaccharide" These include, but are not limited to, "sugars (LPS)," "glycosylates," and "complex carbohydrates." .

[0054] Immunogenic compositions prepared in aprotic solvents (e.g., DMSO) and aqueous conditions Regarding the percentage of serotypes in the remaining polysaccharide-protein conjugates prepared below, When referring to the number of serotypes prepared in an aprotic solvent, it is simply referring to the number of serotypes in the composition. This means that it refers to the number divided by the total number of types.

[0055] As used herein, all ranges of pH, temperature, concentration, etc. are inclusive. For example, the pH range of 5.0 to 9.0 means a pH of 5.0 and a pH of 9.0. Similarly, the temperature range 4 to 25°C includes the outer limits of that range, i.e. This means that temperatures between 4°C and 25°C are included.

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

[0057] In certain embodiments, 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, One or more of 35F and 38 can be prepared by reductive amination in an aprotic solvent. In certain embodiments, serotypes 1, 3, 4, 5, 9V, 11A, 12F and Pneumococcal polysaccharides derived from one or more of the 14 species are reductively amino- Page 14 amino acids in aprotic solvents. In certain embodiments, 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 The derived pneumococcal polysaccharides were prepared using reductive amination in aprotic solvents. In one embodiment, a pneumococcal polyclonal antibody from one or both of serotypes 3 or 18C is Sugars are conjugated to carrier proteins using reductive amination in aprotic solvents Polysaccharides from other serotypes in the multivalent composition may be present in aqueous or aprotic solvents. The conjugation may be carried out using reductive amination in a solvent. Polysaccharides from other serotypes of B. diffractinomycin A can be prepared in aprotic or aqueous solvents. They may be conjugated using chemistry.

[0058] Capsular polysaccharides from Streptococcus pneumoniae are prepared by standard techniques known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and purified by known methods (e.g., See, for example, European Patent Nos. 497524 and 497525), and preferably by microfluidization, achieved using a homogenizer, or by chemical hydrolysis In one embodiment, each lung Streptococcus pneumoniae polysaccharide serovars are grown in soy-based media. Individual polysaccharides are purified by standard processes, including filtration. See, e.g., U.S. Patent Publication No. 2008 See U.S. Patent No. 5,847,112 and U.S. Patent No. 5,847,112. To reduce viscosity and / or use techniques such as mechanical or chemical sizing To improve the filterability of the conjugated product, polysaccharides can be sized. Chemical hydrolysis can be carried out using acetic acid. Mechanical sizing can be carried out using This can be done using high pressure homogenizing shear.

[0059] In some embodiments, the purified polysaccharide prior to conjugation is between 5 kDa and 4,0 The molecular weight was determined by multi-angle light scattering (MALS) and refractive index. Calculated by size exclusion chromatography (SEC) combined with a radioisotope detector (RI). In other such embodiments, the polysaccharide may be between 10 kDa and 4,000 kDa. ;50kDa~4,000kDa;50kDa~3,000kDa;50kDa~2,0 00kDa;50kDa~1,500kDa;50kDa~1,000kDa;50kDa a~750kDa;50kDa~500kDa;100kDa~4,000kDa;10 0kDa~3,000kDa;100kDa~2,000kDa;100kDa~1,5 00kDa;100kDa~1,000kDa;100kDa~750kDa;100kDa Da~500kDa;100~400kDa;200kDa~4,000kDa;200 kDa~3,000kDa;200kDa~2,000kDa;200kDa~1,50 0 kDa; 200 kDa to 1,000 kDa, or 200 kDa to 500 kDa It has a molecular weight.

[0060] The purified polysaccharide is chemically activated to allow the sugar to react with the carrier protein. The purified polysaccharide can be linked to a linker. Once activated or Once connected to the linker, each capsular polysaccharide can be separately conjugated to a carrier protein to form a complex. Polysaccharide conjugates may be prepared by known coupling techniques. good.

[0061] The polysaccharide is coupled to a linker to form a polysaccharide- A linker intermediate can be formed. Thus, the linker has at least one terminal The other end is an ester group, and the linker reacts with the polysaccharide to form a polysaccharide-phosphorus bond. It is selected so as to be capable of forming a Kerr intermediate.

[0062] The polysaccharide can be coupled to a linker using the primary amine groups therein. In this case, the linker typically has an ester group at each end. Coupling is achieved by reacting one of the ester groups with a primary amine group in the polysaccharide via substitution. The reaction allows the polysaccharide to be coupled to the linker via an amide bond. Thus, the linker is the first linker in the polysaccharide. a first ester group for reacting with primary amine groups in the carrier molecule; A typical linker is an azide-based bifunctional linker that provides a second ester group for the linker. Pinic acid N-hydroxysuccinimide diester (SIDEA).

[0063] Coupling can also be done indirectly, i.e., by derivatizing the polysaccharide prior to coupling to the linker. This may also occur with an additional linker being used to conjugate the compound.

[0064] The polysaccharide is coupled to a further linker using the carbonyl group at the reducing end of the polysaccharide. This coupling involves (a1) reacting the carbonyl group with a further linker, and (a 2) reacting the free end of the additional linker with the linker. In this embodiment, the further linker typically has primary amine groups at both ends, React one of the primary amine groups with a carbonyl group in the polysaccharide by reductive amination using This allows the step (a1) to be carried out. A primary amine group is used. A hydrazide or hydroxylamino group is suitable. Primary amine groups are typically present at both ends of the further linker. A polysaccharide-further linker intermediate coupled to a further linker via a C-N bond results.

[0065] The polysaccharides can be coupled to further linkers using different groups therein, particularly carboxyl groups. This coupling can be accomplished by (a1) reacting the group with a 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, thereby Reaction of one of the primary amine groups with a carboxyl group in the polysaccharide by EDAC activation This allows the step (a1) to be carried out. A hydrazide group is suitable. The same primary amine group is used. is typically present at both ends of the further linker. via an amide bond to yield a polysaccharide-further linker intermediate.

[0066] In one embodiment, the carrier is prepared by chemical activation of the polysaccharide followed by reductive amination. Conjugation to proteins is described in U.S. Pat. Nos. 4,365,170 and 4,677,687. Nos. 3,574 and 4,902,506, U.S. Patent Publication No. 2006 / 022838 0, 2007 / 184072, 2007 / 0231340 and 20 07 / 0184071, and WO 2006 / 110381 and WO 2008 by the means described in patent applications 2008 / 079653 and 2008 / 143709 This can be achieved by chemically treating periodates (sodium periodate, potassium periodate, etc.). Oxidizing terminal hydroxyl groups to aldehydes, such as with ammonium or periodate This may require activation of pneumococcal polysaccharides by reaction with an optional oxidizing agent. , random acyl groups of adjacent hydroxyl groups of carbohydrates, forming reactive aldehyde groups. This results in chemical cleavage.

[0067] In one embodiment, the range is 0.01 to 10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7-0.8, 0.05-0.5, 0.1-0.3 molar equivalents of oxidizing agent and polysaccharide In one embodiment, about 0.1, 0.15, 0.2, 0.25, 0.3, 0.3 5, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0. The polysaccharide was reacted with 8, 0.85, 0.9, and 0.95 molar equivalents of oxidizing agent. To achieve activation, a smaller amount of peroxide, e.g., 0.1-0.3 Meq, is used for activation. Use urate (e.g., 0.05-0.22 or 0.05 per mole of polysaccharide repeating unit). It is generally preferred to use a mixture of 0.09 to 0.22 moles of aldehyde. "Activation level" refers to the number of moles of aldehyde per mole of polysaccharide repeating unit. The less polymerization, the more natural the polysaccharide. is converted to aldehyde.

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

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

[0070] Coupling to carrier proteins is achieved by direct amination of the lysyl groups of the protein. For example, a mixture of activated polysaccharide and carrier protein is by reacting with a reducing agent such as sodium cyanoborohydride in the presence of nickel. The conjugation reaction is carried out in aqueous or humid water. This can occur in the presence of dimethyl sulfoxide (DMSO). See, for example, U.S. Patent Publication No. 2015 / 0231270 and 2011 / 0195086 and European Patent No. EP04 See Patent No. 71177B1. Unreacted aldehyde is then removed by cleavage with sodium borohydride. Capping is performed by adding a strong reducing agent such as

[0071] Reductive amination involves (1) oxidizing polysaccharides to form reactive aldehydes and (2) oxidizing activated polysaccharides. The imine (Schiff base) formed between the sugar and the carrier protein is reduced to form a stable amine covalent bond. The oxidation process involves two steps: forming a bond to the polysaccharide and then cleaving it. The polysaccharide may be reduced in size prior to oxidation. Mechanical methods (e.g., homogenization) or chemical hydrolysis may be used. The oxidation step can be carried out using acetic acid. The oxidation step can involve reaction with periodate. For purposes of the invention, the term "periodate" includes both periodate and periodic acid. The term also refers to metaperiodate (IO4 - ) and orthoperiodate (IO6 - ) and various salts of periodate (e.g., sodium periodate and periodic acid In one embodiment, the capsular polysaccharide is preferably lysed in the presence of metaperiodate. Preferably, the oxidation is carried out in the presence of sodium periodate (NaIO). In this case, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid. will be done.

[0072] In one embodiment, the oxidizing agent is present in the presence of an oxidizing agent that selectively oxidizes primary hydroxyls. In the above, stable nitroxyl or nitroxide radical compounds, such as those disclosed in WO 2004 / 024444, piperidine-N-oxy or pyrrolidine- as described in Patent Publication No. 14 / 097099 In the reaction, the actual oxidizing agent is the N-oxy compound. In one embodiment, the stable nitroxy or nitroxyl is a nitroxy or nitroxammonium salt. The pyroxide radical compounds are piperidine-N-oxy or pyrrolidine-N-oxy compounds. In one embodiment, the stable nitroxyl or nitroxide radical The compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or has a PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety In one embodiment, the stable nitroxyl radical compound is TEMPO or its derivatives. In one embodiment, the oxidizing agent is a molecule having an N-halo moiety. In one embodiment, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide Imide, 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, diiodoisocyanate Annuric Acid and 1,3,5-Triiodo-1,3,5-triazinane-2,4,6-tri On is selected from the group consisting of.

[0073] In certain embodiments, the oxidizing agent is a hydroxybenzoate (as described in WO 2014 / 097099). 2,2,6,6-Tetramethyl-1-piperidinyloxy (T) as a co-oxidant EMPO) free radical and N-chlorosuccinimide (NCS). In one embodiment, the glycoconjugates derived from Streptococcus pneumoniae are: a) 2,2,6 in an aqueous medium; ,6-Tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccine a) reacting a sugar with an imide (NCS) to produce an activated sugar; and b) reacting the activated sugar with one or more (hereinafter, The method is referred to as "TEMPO / NCS-reductive amination").

[0074] The oxidation reaction may be quenched by the addition of a quenching agent. The quenching agent may be a vicinal diol, 1, 2-amino alcohol, amino acid, glutathione, sulfite, bisulfite, dithionite , metabisulfite, thiosulfate, phosphite, hypophosphite or phosphorous acid (glycerol ethylene glycol, propane-1,2-diol, butane-1,2-diol or may be selected from butane-2,3-diol, ascorbic acid, etc.

[0075] The second step in the conjugation process involves the use of a reducing agent to bind the activated polysaccharide to the carrier protein. Reduction of imine (Schiff base) bonds with proteins to form stable conjugate bonds (so-called reductive amination). Suitable reducing agents include cyanoborohydride (cyanoborohydride). In one embodiment, the hydroxybenzoates include sodium borohydride or sodium borohydride. In this case, the reducing agent is sodium cyanoborohydride.

[0076] In certain embodiments of the method of the present invention, the reductive amination reaction is carried out in an aprotic solvent (or In one embodiment, the reduction reaction is carried out in DMSO. In the case of freeze-drying, the process is carried out in a solvent such as dimethylformamide (DMF). Activated polysaccharides and carrier proteins can be reconstituted using DMSO or DMF solvents. 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. and can be capped with a suitable capping agent. In the present invention, the capping agent is sodium borohydride (NaBH4). Alternatively, triacetoxyhydrogenation in the presence of a Bronsted or Lewis acid is used. Sodium borohydride or sodium or zinc borohydride, pyridine borane, 2-pico Phosphorus borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe' PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane amine boranes such as PEMB, or borohydride exchange resins. Following gating (reduction and optional capping), the glycoconjugates can be purified by a method known in the art. The polysaccharide-protein conjugates may be purified by various techniques known in the art (e.g., by the use of a saccharide-protein conjugate). These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration, filtration, precipitation / elution, column chromatography (ion exchange chromatography, multimodal chromatography) Dual ion exchange chromatography, DEAE or hydrophobic interaction chromatography In one embodiment, the glycoconjugates are purified by diafiltration or infiltration. Purified by ion-exchange or size-exclusion chromatography .

[0078] Glycoconjugates prepared using reductive amination in aprotic solvents generally have multivalent phospholipids. It is used in the Streptococcus pneumoniae conjugate vaccine. Therefore, all serotypes are non-protease. In certain embodiments for polyvalent compositions that are not necessarily prepared in ionic solvents, The reduction reaction of these serotypes is carried out in aqueous media (e.g., pH 6.0-8.5, 7.0-8.0, or or 7.0-7.5, PBS (phosphate buffered saline), MES (2-(N-morpholino) (4-(2-hydroxyethyl)-1-piperazine), ethanesulfonic acid, HEPES, ethanesulfonic acid), Bis-Tris, ADA (N-(2-acetamido)iminodiacetic acid ), PIPES (piperazine-N,N')-bis(2-ethanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES(N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino) )) propanesulfonic acid), DIPSO (3-bis(2-hydroxyethyl)amino-2 -hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butane sulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N-(2-hydroxyethyl) hydroxypropanesulfonic acid, POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid) -3-propanesulfonic acid), TEA (triethanolamine), EPPS(4-(2 -hydroxyethyl)piperazine-1-propanesulfonic acid), bicine or HEPB The study is carried out in a

[0079] In some embodiments, the glycoconjugates of the present invention are between 10 kDa and 10,000 kDa. In other such embodiments, the polysaccharide comprises a polysaccharide having a molecular weight of 25 kDa to 5,0 In other such embodiments, the polysaccharide has a molecular weight of 50 kDa to 1,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 70 kDa and 900 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 100 kDa and 800 kDa. In other such embodiments, the polysaccharide has a molecular weight of 200 kDa to 600 kDa. In further such embodiments, the polysaccharide has a molecular weight of 100 kDa to 1000 kDa. kDa;100kDa~900kDa;100kDa~800kDa;100kDa~7 00kDa;100kDa~600kDa;100kDa~500kDa;100kDa ~400kDa;100kDa~300kDa;150kDa~1,000kDa;15 0kDa~900kDa;150kDa~800kDa;150kDa~700kDa; 150kDa~600kDa;150kDa~500kDa;150kDa~400kDa a;150kDa~300kDa;200kDa~1,000kDa;200kDa~9 00kDa;200kDa~800kDa;200kDa~700kDa;200kDa ~600kDa;200kDa~500kDa;200kDa~400kDa;200k Da~300;250kDa~1,000kDa;250kDa~900kDa;250 kDa~800kDa;250kDa~700kDa;250kDa~600kDa;2 50kDa~500kDa;250kDa~400kDa;250kDa~350kDa ;300kDa~1,000kDa;300kDa~900kDa;300kDa~80 0kDa;300kDa~700kDa;300kDa~600kDa;300kDa~ 500kDa;300kDa~400kDa;400kDa~1,000kDa;400 kDa~900kDa;400kDa~800kDa;400kDa~700kDa;4 00kDa to 600kDa; 500kDa to 600kDa.

[0080] In some embodiments, the glycoconjugates of the present invention have a molecular weight of 1,000 kDa to 10,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of 1,000 kDa to 7, In other such embodiments, the polysaccharide has a molecular weight of 1,000 kDa. It has a molecular weight of ~6,000 kDa.

[0081] In certain embodiments, the conjugation reaction is carried out by reductive amination, Here, nickel is used for greater conjugation reaction efficiency and for the reduction of free cyanide. Transition metals can form stable complexes with cyanides. It is known that sodium cyanoborohydride is used to remove amino groups and It is known to improve the reductive methylation of formaldehyde (S. Gidley et al.,Biochem J.1982,203:331-334;Jentof Anal Biochem. 1980, 106:186-190). Residue The addition of nickel inhibits the formation of cyanide by complexing the inhibitory cyanide that remains during conjugation. This increases protein consumption and leads to larger, potentially more immunogenic conjugates. This results in the formation of

[0082] Differences in starting cyanide levels among sodium cyanoborohydride reagent lots also Conjugation performance can vary, resulting in variations in conjugate size and conjugate size. Gate Ps vs CRM 197 The addition of nickel changes the product properties such as the cyanide ratio. Complexation of the cyanoborohydride reduces conjugation variability. Differences between sodium lots have been eliminated.

[0083] A suitable alternative chemistry is 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. This involves activating sugars with cyanate (CDAP) to form cyanate esters. Thus, the activated sugar can be attached to a carrier protein directly or via a spacer (linker) group. For example, the spacer may be cystamine or Cysteamine can give rise to thiolated polysaccharides, which can be imide-activated carrier proteins (e.g., with GMBS) or haloacetylated carriers Proteins (e.g., iodoacetimide [e.g., ethyl iodoacetimide HCl] or N-Succinimidyl Bromoacetate or SIAB or SIA or SBAP It can be coupled to the support via a thioether bond obtained after reaction with For example, a cyanate ester (which may be prepared by CDAP chemistry) may be mixed with hexanediamine. Coupling with amines or adipic acid dihydrazide (ADH) onto protein supports using carbodiimide (e.g., EDAC or EDC) chemistry via the carboxyl group of The amino-derivatized sugar is conjugated to a carrier protein. are disclosed in International Publication Nos. WO 93 / 15760, WO 95 / 08348 and WO 96 / 2909 No. 4, and Chu et al., 1983, Infect. Immunity 4 0:245-256.

[0084] Other suitable techniques include carbodiimides, hydrazides, active esters, norborane, p-dinitrobenzoates, Uses benzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in WO 98 / 42721. The carbonyl linker may also include a carbonyl linker that is connected to a free hydroxyl group of the sugar. The group was reacted with CDI (Bethell et al., 1979, J. Biol. C hem.254:2572-4;Hearn et al.,1981,J.Chrom atogr.218:509-18), and then reacting with proteins. This may be achieved by forming a carbamate bond at the anomeric end of the reduction to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, The reaction of the alkyloxyl group with CDI to form a CDI carbamate intermediate, and the formation of CDI carbamate It may involve coupling of a bamate intermediate with an amino group on the protein.

[0085] After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugate is The conjugate is purified by one or more of a variety of techniques (polysaccharide-protein conjugates). 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 US Patent No. 6,146,902.

[0086] Another method for characterizing the glycoconjugates of the present invention is to characterize the carrier that becomes conjugated to the sugar. Proteins (e.g., CRM 197 ) is due to the number of lysine residues in The degree of conjugation can be characterized as the extent of conjugation of the lysine. Evidence for lysine modification of carrier proteins by covalent attachment to sugars has been reported in the literature, including studies known to those skilled in the art. This can be obtained by amino acid analysis using conventional methods. Recovery compared to the carrier protein starting material used to generate the conjugate material In one embodiment, the conjugates of the glycoconjugates of the present invention are The degrees of rotation are 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 10 to 18, 7 to 13, 8 to 18, 8 to 13, 10 to 18, or 10 to 13. In some embodiments, the degree of conjugation of the glycoconjugates of the present 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 present invention is 7 to 18. In some such embodiments, the carrier protein is a CRM 197 is.

[0087] The glycoconjugates of the present invention may also be characterized by the sugar to carrier protein ratio (wt / wt). In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate may be (w / w) is 0.5 to 3.0 (approx. 0.5, approx. 0.6, approx. 0.7, approx. 0.8, approx. 0.9, approx. 1.0, approx. 1.1, approx. 1.2, approx. 1.3, approx. 1.4, approx. 1.5, approx. 1.6, approx. 1.7, approx. 1.8, approx. 1.9, approx. 2.0, approx. 2.1, approx. 2.2, approx. 2.3, approx. 2.4, approx. 2.5, approx. In other embodiments, the β-glucan content is about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0. In this case, the sugar to carrier protein ratio (w / w) was 0.5-2.0, 0.5-1.5, 0. 8 to 1.2, 0.5 to 1.0, 1.0 to 1.5, or 1.0 to 2.0. In one embodiment, the sugar to carrier protein ratio (w / w) is 0.8 to 1.2. wherein the ratio of capsular polysaccharide to carrier protein in the conjugate is 0.9 to 1.1 In some such embodiments, the carrier protein is a CRM 197 The complex of the present invention The carbohydrate and immunogenic compositions contain free sugars that are not covalently attached to a carrier protein. Free sugars may be present in the glycoconjugate composition but are still non-covalently attached to the glycoconjugate. It may be associated (i.e., non-covalently bound, adsorbed, or entrapped in or with) stomach.

[0088] In one embodiment, the complex carbohydrates comprise about 50%, 45%, 40% or more of the total amount of polysaccharides. In one embodiment, the composition contains less than 35%, 30%, 25%, 20% or 15% free 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 this form, the complex carbohydrate contains less than about 15% free polysaccharides compared to the total amount of polysaccharides.

[0089] Multivalent polysaccharide-protein conjugate vaccines The multivalent pneumococcal immunogenic composition comprises one or more pneumococcal antibodies conjugated to one or more carrier proteins. , 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10 A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A , 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28 Choose at least one of A, 31, 33F, 34, 35A, 35B, 35F and 38 and the capsular polysaccharides derived from at least one serotype of Streptococcus pneumoniae. Polysaccharides derived from the type were prepared using reductive amination in aprotic solvents such as DMSO. The present invention provides at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, Due to serotypes 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 The present invention contemplates multivalent pneumococcal immunogenic compositions having polysaccharides of the following nature. The saccharide derived from is not conjugated to multiple carrier proteins.

[0090] In certain embodiments, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 blood Polysaccharides derived from the Qing type were synthesized using reductive amination in aprotic solvents such as DMSO. It is prepared.

[0091] In particular embodiments, serotypes 3, 6A, 6B, 7F, 18C, 19A, 19F or One or more of the 23Fs were prepared using reductive amination in aprotic solvents. In a particular aspect of this embodiment, one or both of serotypes 3 or 18C are non- It is prepared using reductive amination in a protic solvent.

[0092] In certain embodiments, at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% are non-pro The remaining serotypes are prepared using alternative chemistries and / or in aqueous solutions. It is prepared in a solvent.

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

[0094] In one embodiment, the multivalent composition is prepared by the reductive activating of DMSO in an aprotic solvent. Serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23 prepared using immunofluorescence Polysaccharides derived from F and serotypes 1 and 3 prepared using reductive amination in aqueous media , 4, 5, 9V, 14, 22F and 33F.

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

[0096] Carrier proteins In certain embodiments of the present invention, the CRM 197 is used as a carrier protein. RM 197 is a non-toxic variant of diphtheria toxin (i.e., a toxoid). In terms of form, CRM 197 was grown in casamino acid and yeast extract-based media. Corynebacterium diphtheria strain C7(β1 97). In another embodiment, the CRM 197 is a U.S. Pat. It is typically prepared recombinantly according to the methods described in CRM 19 7 is a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, the CRM is purified by 197 Pfenex Expression Technology™ (Pfenex, Inc., California) Pseudomonas fluorescens (Pseudomonas fluorescens) was used. It is prepared in Bacillus subtilis (Bacillus fluorescens).

[0097] Other suitable carrier proteins include DT (diphtheria toxoid) or DT (DT FB) fragment B, TT (tetanus toxoid) or TT fragment C, pertussis toxoid, Leratoxoids (e.g., as described in WO 2004 / 083251) E. coli LT, E. coli ST, and Pseudomonas aeruginosa Further inactivating bacterial toxins include exotoxin A from Bacterial outer membrane proteins. Proteins, e.g., outer membrane complex c (OMPC), porins, transferrin-binding proteins protein, pneumococcal surface protein A (PspA; see WO 02 / 091998). ), pneumococcal adsorption protein (PsaA), C5a peptide from group A or B streptococci putidase, or Haemophilus influenzae ) protein D, pneumolysin of Streptococcus pneumoniae (Kuo et al., 1995, Inf ect Immun 63;2706-13) (detoxified in some way, e.g. For example, dPLY-GMBS (see WO 04 / 081515) or dPLY -formol), PhtX(PhtA, PhtB, PhtD, PhtE and Pht Protein fusions, such as PhtDE fusions, PhtBE fusions (WO 01 / 999). 8334 and 03 / 54007)) can also be used. Proteins, e.g., ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin BSA or purified protein derivative of tuberculin (PPD), PorB (myelin meningitidis), PD (protein D of Haemophilus influenzae; e.g., EP 05946 10B), or their immunologically functional equivalents, synthetic peptides (see European Patent No. 03788 81 and 0427347), heat shock proteins (see WO 93 / 1 7712 and 94 / 03208), pertussis proteins (see WO 98 / 03208). 58668 and EP 0471177), cytokines, lymphokines, growth factors or hormones (see WO 91 / 01146), various pathogen-derived antibodies An artificial protein (Falug) containing multiple human CD4+ T cell epitopes derived from a source i et al.,2001,Eur J Immunol 31:3816-3824 ), e.g., the N19 protein (Baraldoi et al., 2004, I Infect Immun 72:4884-7), iron uptake protein (International Publication 01 / 72337), C. difficile toxin A or B (see International Publication No. No. 00 / 61761), and flagellin (Ben-Yedidia et al. al., 1998, Immunol Lett 64:9) also serves as a carrier protein. It can be used.

[0098] Other DT variants, e.g., CRM 176 , CRM 228 , CRM 45 (Uchida et al., 1973, J Biol Chem 218:3838-3844);C RM9, CRM 45 , CRM 102 , CRM 103 and CRM 107 , and Nic Genetically Engineered by Holls and Youle Toxins, Ed: Frankel, Maecel Dekker Inc, 1992 Glu-148 to Asp, Gln, or Ser; and and / or deletion or mutation of Ala158 to Gly, as well as U.S. Pat. No. 4,709, or other mutations disclosed in US Pat. No. 4,950,740; residues Lys516, Ly At least one mutation at s526, Phe530, and / or Lys534, if and other variations disclosed in U.S. Pat. Nos. 5,917,017 or 6,455,673. or a fragment disclosed in U.S. Pat. No. 5,843,711, attached to a second carrier protein. Such DT mutants can also be used to generate DT containing B fragments. The FB variant can contain an epitope region.

[0099] When a multivalent vaccine is used, a second antibody may be administered to one or more of the antigens in the multivalent vaccine. A carrier can be used. The second carrier protein is preferably non-toxic and non-reactive. The second carrier protein is also a protein that is of a high affinity and is obtained in sufficient quantity and purity. Also, to enhance the immunogenicity of the antigen, the antigen may be conjugated to, for example, Streptococcus pneumoniae polysaccharide. Carrier proteins should follow standard conjugation procedures. In one embodiment, each pod that is not conjugated to a first carrier protein The membrane polysaccharides are conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule are conjugated to a single carrier protein). The unconjugated capsular polysaccharide is conjugated to two or more carrier proteins. conjugated (each capsular polysaccharide molecule is conjugated to a single carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically bound to the same carrier protein. It is conjugated.

[0100] Pharmaceutical / vaccine compositions The present invention further provides the above polysaccharide serotypes together with a pharmaceutically acceptable carrier and adjuvant. A pharmaceutical composition comprising, consisting essentially of, or consisting of any of the combinations of Compositions, including products, immunogenic compositions and vaccine compositions, are further provided.

[0101] The formulation of the polysaccharide-protein conjugates of the present invention is art-recognized. For example, individual pneumococcal conjugates can be prepared by the method described above. A physiologically acceptable vehicle can be added to prepare the composition. Examples of vehicles include water, buffered saline, polyols (e.g., glycerol, propyl polyethylene glycol, liquid polyethylene glycol) and dextrose solution. However, the present invention is not limited to these.

[0102] In one embodiment, the vaccine composition comprises an L-histidine buffer containing sodium chloride. It is compounded by

[0103] As defined herein, an "adjuvant" is an agent that acts as an immunogen in the immunogenic composition of the invention. Immune adjuvants are substances that help enhance the immunogenicity of a given vaccine when administered alone. enhances immune responses to weak antigens, e.g., no or weak antibody titers or cellular Induce an immune response, increase antibody titers to an antigen, and / or enhance an immune response in an individual Therefore, adjuvants can reduce the effective dose of antigen to be achieved. These are often administered to enhance the immune response and are well known to those skilled in the art. Suitable adjuvants for enhancing the effect include, but are not limited to: do not have.

[0104] (1) Aluminum salts (alum), such as aluminum hydroxide and aluminum phosphate Um, aluminum sulfate; (2) an oil-in-water emulsion formulation (muramyl peptide (defined below) or bacterial cell wall (a) 5% squalane, with or without other specific immunostimulants such as squalane, 0.5% Tween 80 and 0.5% Span 85 (various amounts of MTP -PE), Model 110Y Microfluidizer (Micr Microfluidizers such as those from Fluidics, Newton, Massachusetts MF59 formulated into submicron particles using (WO 90 / 14837); ) 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L1 21 and thr-MDP, microfluidized into a submicron emulsion. SAF, which is either mixed or vortexed to produce a larger particle size emulsion; (c) 2% Squalene, 0.2% Tween 80, and as described in U.S. Pat. No. 4,912,094 3-O-deacylated monophosphoryl lipid A (MPL™), trehalose diglyceride Detoxifying agent (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox Ribi™ adjuvants containing one or more bacterial cell wall components from the group consisting of The RAS (Corixa, Hamilton, MA); and )Montanide ISA; (3) Saponin adjuvants, such as Quil A or STIMULON (trademark) ) QS-21 (Antigenics, Framingham, MA) (e.g. (See U.S. Patent No. 5,057,540) may be used, or this adjuvant particles produced from, for example, ISCOMs (cholesterol, saponin, phospholipids and Immunostimulating complexes (Immunostimulating complexes formed by the combination of amphipathic proteins) and Iscom atrix® (which has essentially the same structure as ISCOM but contains the protein) not); (4) Bacterial lipopolysaccharides, synthetic lipid A analogs, e.g., aminoalkylglucosamine phosphate Antioxidant phosphate (AGP), or a derivative or analog thereof, available from Corixa and is described in U.S. Pat. No. 6,113,918; one such AGP is a 2- [(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl 2-deoxy- 4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl] -2-[(R)-3-tetradecanoyloxytetradecanoylamino]-bD-glucan copyranoside, also known as 529 (formerly known as RC529) (previously known as "antioxidant"), formulated in aqueous form or as a stable emulsion; (5) Synthetic polynucleotides, e.g., containing CpG motif(s). oligonucleotides (U.S. Patent 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), Inter -ferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor ( GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TN F), costimulatory molecules B7-1 and B7-2; (7) Complement, e.g., the trimer of complement component C3d.

[0105] In another embodiment, the adjuvant is a combination of two, three or more of the above adjuvants. a mixture of, for example, SBAS2 (3-deacylated monophosphoryl lipid A and QS21) It is an oil-in-water emulsion containing

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

[0107] In certain embodiments, the adjuvant is an aluminum salt. The vaccine may be an alum-precipitated or alum-adsorbed vaccine. Sodium salt adjuvants are well known in the art and are described, for example, in Harlow, E. and and D. Lane (1988; Antibodies: A Laboratory Ma National Cold Spring Harbor Laboratory) and N icklas, W. (1992;Aluminum salts.Research i In Immunology 143:489-493. The salts include hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum Aluminum hydrate, aluminum trihydrate, hydrogel, Superfos, Amphogel, Aluminum hydroxide (III), aluminum sulfate hydroxyphosphate, aluminum phosphate adjuvant (APA), amorphous alumina, alumina trihydrate, or trihydroxyalumina These include, but are not limited to, aluminum.

[0108] APA is an aqueous suspension of aluminum hydroxyphosphate. APA is also known as aluminum chloride. A 1:1 volumetric blend of aluminum and sodium phosphate to form aluminum hydroxyphosphate. After the blending process, the material is mixed with a high shear mixer. The product is then reduced in size in a furnace to achieve a monodisperse particle size distribution. Diafilter and steam sterilize.

[0109] In certain embodiments, commercially available Al(OH) (e.g., from West Virginia, NY) Denmark / Accurate Chemical and Scien in Lee Protein The protein is adsorbed at a ratio of 50-200g protein / mg aluminum hydroxide. In another embodiment, protein adsorption depends on the pI (isoelectric pH) of the protein and the pH of the medium. Proteins with low pI have a stronger positive aluminum charge than proteins with high pI. The aluminum salts are adsorbed onto the antigen, which is slowly released over a period of 2-3 weeks. establish a reservoir of IgG, participate in nonspecific activation of macrophages and complement activation, and / or Or it may stimulate the innate immune system (possibly through stimulation of uric acid). See echt et al., 2009, Curr Opin Immunol 21:23 .

[0110] Monovalent bulk aqueous conjugates are typically blended together to achieve a target concentration of 16 μg / mL. All serotypes were diluted to a target of 8 μg / mL except for 6B, which was diluted to 8 μg / mL. The batch is then filter sterilized and an equal volume of aluminum phosphate adjuvant is added to the desired final volume. Add aluminum aseptically to a concentration of 250 μg / mL. The batch will be filled into single-use 0.5 mL / dose vials.

[0111] In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, e.g., C pG-containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG In another embodiment, the adjuvant is a Coley Pharma The product is ODN1826 available from the European Medicines Agency.

[0112] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "CpG nucleotide" and similar terms refer to a nucleotide sequence of 6 to 50 nucleotides containing an unmethylated CpG moiety. For example, Wang et al., 2003, Vacc See, e.g., ine 21:4297. In another embodiment, any other art-specific definition of this term is used. The definition accepted in the field is intended. A CpG-containing oligonucleotide is any synthetic nucleotide. Modified oligonucleotides include those using interosidic linkages, modified bases and / or modified sugars.

[0113] Methods for using CpG oligonucleotides are well known in the art, see, for example, Su r et al.,1999,J Immunol.162:6284-93;Vert helyi,2006,Methods Mol Med.127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug C Arrier Syst.23:89-110.

[0114] Administration / Dosage The compositions and formulations of the present invention allow for the administration of vaccines by systemic or mucosal routes. and to protect or treat humans susceptible to infection, e.g., pneumococcal infection, by In one embodiment, the present invention provides an immunologically effective amount of an immunogen of the present invention. and administering to a human an active ingredient composition for the treatment of Streptococcus pneumoniae capsular polysaccharide conjugates. In another embodiment, the present invention provides a method for inducing an immune response, comprising administering an immunologically effective amount of the present invention to a patient. A method for vaccinating a human against pneumococcal infection, comprising administering to the human an immunogenic composition of the invention. The present invention provides a method for inoculating a vaccine.

[0115] The optimal amount of components for a particular vaccine can be determined through standard clinical trials involving the observation of an appropriate immune response in subjects. For example, in another embodiment, the vaccine can be administered to humans. Dosages for vaccination are determined by extrapolating animal studies to human data. In another embodiment, the dosage is empirically determined. It has been demonstrated to be immunogenic in animal data.

[0116] An "effective amount" of the composition of the present invention is an amount that prevents infection with a microorganism, e.g., Streptococcus pneumoniae, during a subsequent attack. The term "dose" refers to the dose required to induce antibodies that significantly reduce the likelihood or severity of a disease.

[0117] The method of the present invention is applicable to the treatment of invasive infections (meningitis, pneumonia, and bacteremia) and non-invasive infections (acute middle ear infections). caused by microorganisms, such as Streptococcus pneumoniae, including both rhinosinusitis and sinusitis It can be used to prevent and / or alleviate major clinical symptoms.

[0118] Administration of the compositions of the invention may be by injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; or The administration may include one or more of mucosal administration to the oral / alimentary tract, the respiratory tract or the genitourinary tract. In some cases, intranasal administration is used to treat pneumonia or otitis media (nasopharyngeal infection caused by pneumococcal To more effectively prevent carriage and thereby reduce infection at the earliest stage ).

[0119] The amount of conjugate in each vaccine dose was determined to be sufficient to induce an immune protective response without significant adverse effects. Such amounts may vary depending on the serotype of pneumococcus. For polysaccharide-based conjugates, each dose ranges from 0.1 to 100 μg of each polysaccharide. , particularly 0.1 to 10 μg, more particularly 1 to 5 μg. For example, each dose may contain 100 , 150, 200, 250, 300, 400, 500, or 750ng 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 10 It may contain 0 μg.

[0120] The optimal amount of components for a particular vaccine can be determined through standard clinical trials involving the observation of an appropriate immune response in subjects. For example, in another embodiment, the vaccine can be administered to humans. Dosages for vaccination are determined by extrapolating animal studies to human data. In another embodiment, the dosage is empirically determined.

[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 In yet another embodiment, the above-mentioned microcrystalline cellulose is 1, 1.2, 1.5, 2, 3, 5 mg or more. The dose of alum salt is given 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 (under 1 year of age), a toddler (about 12-24 months of age), or In other embodiments, the human subject is an elderly subject (e.g., For example, over 50 or over 65 years of age. The compositions of the present invention are also suitable for older children, It is also suitable for use in adolescents and adults (e.g., 18-45 years or 18-65 years).

[0123] In one embodiment of the method of the present invention, the composition of the present invention is administered as a single inoculation. In another embodiment, the vaccine is administered two, three, four or more times, well spaced apart. For example, the composition may be administered at 1, 2, 3, 4, 5, or 6 month intervals, or The immunization schedule may be administered at intervals of 10-20 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 120 minutes, 140 minutes, 160 minutes, 180 minutes, 200 minutes, 220 minutes, 240 minutes, 280 minutes, 300 minutes, 3 The schedule may be according to that specified for cocci vaccines. For example, Streptococcus pneumoniae The routine schedule for infants and young children for invasive diseases caused by bacteria is , at ages 2, 4, 6, and 12 to 15 months. The composition is administered as a series of four doses at ages 2, 4, 6, and 12-15 months. will be done.

[0124] The compositions of the present invention may also comprise one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those described in WO 02 / 0838 55 and 02 / 053761.

[0125] compound The compositions of the present invention can be administered parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, or intradermally. The drug is administered to the subject by one or more methods known to those skilled in the art, such as intravenously, intraperitoneally, or the like, It can be formulated according to the

[0126] In one embodiment, the compositions of the present invention are administered by epidermal injection, intramuscular injection, intravenous injection, or intravenous injection of a liquid formulation. It is administered by intra-arterial, subcutaneous, or intra-mucosal injection into the respiratory tract. This includes liquids.

[0127] The compositions of the present invention may be provided as single dose vials, multi-dose vials, or pre-filled. It can be formulated as a dispensing syringe.

[0128] In another embodiment, the compositions of the present invention are administered orally, and therefore in a form suitable for oral administration. Solid oral formulations include tablets, capsules, and capsules. Examples of liquid oral formulations include capsules, pills, granules, and pellets. , suspensions, dispersions, emulsions, oil solutions, etc.

[0129] Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solutions, suspensions, emulsions, and the like. or oil. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol, Cole / water solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, olive oil, The fats may be corn oil, sunflower oil, fish liver oil, another marine oil, or fats from milk or eggs.

[0130] Pharmaceutical compositions may be isotonic, hypotonic or hypertonic. It is often preferred that pharmaceutical compositions for injection be essentially isotonic at the time of administration. Therefore, for storage purposes, the pharmaceutical composition may preferably be isotonic or hypertonic. If the composition is hypertonic for storage, it should be diluted to an isotonic solution before administration.

[0131] The isotonic 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, CaCl2, KCl, and Mg Examples of non-ionic tonicity agents include, but are not limited to, mannitol, methyl ... Examples of suitable glycerols include, but are not limited to, ethanol, sorbitol and glycerol.

[0132] It is also preferred that the at least one pharmaceutically acceptable excipient is a buffer. For some purposes, for example, when the pharmaceutical composition is for infusion or injection, the composition It is often desirable to include a buffer in the product, which can treat the solution to a pH of 4. It can be 10, for example 5 to 9 (6 to 8, etc.).

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

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

[0135] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution. solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution and Intravenous vehicles include fluid and nutrient replenishers, Ringer's dextromethorphan, and fixed oils. Examples include surfactants and electrolyte supplements, such as sucrose-based ones. and other pharmaceutically acceptable adjuvants, such as water or oil. Generally, water, saline, aqueous dextrose and related sugar solutions are used. , glycols such as propylene glycol or polyethylene glycol, polysorbate Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and Poloxamer 188 (P188) is a preferred liquid carrier, especially for injections. Examples of oils include animal and vegetable oils. of natural or synthetic origin, e.g., peanut oil, soybean oil, olive oil, sunflower oil, fish liver It can be oil, another marine oil, or fat from milk or eggs.

[0136] The formulations of the present invention may also contain surfactants. sorbitan ester surfactants (commonly called Tween), especially PS-20 and and PS-80; ethylene oxide (E), sold under the trade name DOWFAX™ O), copolymers of propylene oxide (PO) and / or butylene oxide (BO) - e.g., linear EO / PO block copolymers; octoxynol, which is a repeating ethoxy (oxy-1,2-ethanediyl) groups, and octoxynol-9 (Triton X -100, or t-octylphenoxypolyethoxyethanol) are of particular interest; ( Octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-4 0); phospholipids such as phosphatidylcholine (lecithin); Tergitol™ N P series nonylphenol ethoxylates; triethylene glycol monolaurate Lauryl, cetyl, stearyl and oleyl ethers (Brij 30) Polyoxyethylene fats derived from alcohol (known as Brij surfactants) aromatic ethers; and sorbitan trioleate (Span 85) and sorbitan monooleate sorbitan esters such as laurate (commonly known as SPAN) A preferred surfactant for inclusion in the emulsion is PS-80. is.

[0137] Mixtures of surfactants can be used, such as a PS-80 / Span 85 mixture. Polyoxyethylene such as polyoxyethylene sorbitan monooleate (PS-80) Sorbitan ester and t-octylphenoxypolyethoxyethanol (Triton Combinations of octoxynol compounds such as X-100 are also suitable. The combination contains laureth 9, polyoxyethylene sorbitan ester and / or octoki and cinol.

[0138] Preferred amounts (by weight) of surfactants are as follows: polyoxyethylene sorbitan 0.01 to 1%, especially about 0.1%, of octyl- or nonyl-esters (e.g., PS-80) Nylphenoxypolyoxyethanol (Triton X-100 or Triton series) Other detergents, etc.) 0.001-0.1%, especially 0.005-0.02%. Polyoxyethylene Polyethers (e.g., laureth 9) 0.1 to 20%, preferably 0.1 to 10%, especially 0. 1-1% or about 0.5%.

[0139] In certain embodiments, the composition comprises histidine (20 mM), saline (150 mM), M), and 0.02% PS-20 or 0.04% PS-80 at pH 5.8, and 250 and μg / mL APA (aluminum phosphate adjuvant). 0 is PS-20 or higher in formulation-controlled flocculation during simulated manufacturing and shipping using primary packaging. or in the presence of PS-80, the concentration may range from 0.005% to 0.1% (w / v). The assay consists of histidine, saline, and up to 24 sera in PS-20 or PS-80. The blends of the types are combined and then the blended material is sterilized with or without an antimicrobial preservative. with APA and saline.

[0140] The choice of surfactant may need to be optimized for different drug products and drug substances.1 For multivalent vaccines with 5 or more serotypes, PS-20 and P188 are preferred. The choice of chemistry used to prepare the conjugate also plays a role in the stabilization of the formulation. In particular, different polysaccharide-protein conjugates in a multivalent composition can play an important role. The conjugation reaction used to prepare the compound was carried out in both aqueous and DMSO solvents. We found that the specific surfactant system made a significant difference in stability when using poly The improved stability of sorbate-protein conjugates was greater than that of polysorbate 20 alone. , or poloxamer 188 in combination with polyols.

[0141] The exact mechanisms by which certain detergents protect biological agents are poorly understood. Possible stabilization mechanisms include preferential hydration, Preferential exclusion, competition for the air / liquid interface between the biological agent and the surface, surface tension, and / or Direct interaction of surfactants with biological agents to mask hydrophobic patches that act as aggregation seeds or This includes cases where

[0142] Poloxamers may also be used in the compositions of the present invention. Poloxamers are polyoxyethylene Polyoxypropylene with two adjacent hydrophilic chains of ethylene (poly(ethylene oxide)) A nonionic triblock copolymer consisting of a central hydrophobic chain of propylene (poly(propylene oxide)). Poloxamers are also known under the trade name Pluronic®. The length of the polymer block can be customized, allowing for slightly different There are many different poloxamers with different properties. These copolymers are generally designated by the letter "P" (for poloxamer) followed by It is expressed as a three-digit number, and the first two digits multiplied by 100 is the approximate size of the polyoxypropylene core. The molecular mass is indicated. The last digit multiplied by 10 is the percentage of polyoxyethylene content. (e.g., P407 = polyoxypropylene molecular mass of 4,000 g / mol and and poloxamer with 70% polyoxyethylene content). Trade name: Pluronic In the case of ®, these copolymer coatings have the physical form at room temperature Start with a defining letter (L = liquid, P = paste, F = flake (solid)) and two digits Or followed by three digits. The first digit of the numeric display (two digits of the three digits) Multiplying this by 300 gives the approximate molecular weight of the hydrophobic substance, and multiplying the last digit by 10 indicates the percentage of polyoxyethylene content (e.g., L61 = 1,800 g / mol polyoxypropylene molecular mass and 10% polyoxyethylene content Pluronic® (see U.S. Patent No. 3,740,421).

[0143] Examples of poloxamers have the following general formula: HO(C2H4O) a (C3H6O) b (C2H4O) 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 of 1100 to 17,400 Da, 7,500 to 15,000 Da. ,000 Da, or in the range of 7,500 to 10,000 Da. The poloxamer may be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulation is 0.001% to 5% weight / volume, or 0.025% to 1% weight / volume. In certain embodiments, the polyol is propylene glycol. In a particular embodiment, the polyol is polyethylene. The solution is ethylene glycol 400, at a final concentration of 1% to 20% weight / volume.

[0145] Suitable polyols for the formulations of the present invention are polymer polyols, especially propylene glycol polyols. polyethylene glycol, polyethylene glycol monomethyl ether Examples of polyether diols include, but are not limited to, propylene glycol, which has a viscosity of about 42 Available in a range of monomer molecular weights from 5 to 2700. Polyethylene glycol and polyethylene glycol monomethyl ethers, also known as PEG200, PEG300 , PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEG MME 4000 Available in molecular weight ranges ranging from (but not limited to) ~200 to ~35,000 The preferred polyethylene glycol is polyethylene glycol 400. The final concentration of polyol in the formulation of the present invention is 1% to 20% weight / volume or 6% to 20% weight / volume. May be % weight / volume.

[0146] The formulation also contains a pH buffered saline solution. Buffers include, for example, TRIS, acetic acid, Salt, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate Salt, glycinate, histidine, glycine, succinate, HEPES(4-(2-hydroxybenzoate) (N-dimethylethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino) propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and tri The buffer may be selected from the group consisting of ethanolamine buffers. can be adjusted to a pH range of 4-10, 5.2-7.5, or 5.8-7.0 In one embodiment of the present invention, the buffer is a phosphate, succinate, histidine, ME S, MOPS, HEPES, acetate or citrate. The buffer may be, for example, a US solution for parenteral use, particularly if the pharmaceutical formulation is for parenteral use. The buffer concentration may be selected from a P-compatible buffer. In certain embodiments, the buffer may have a final concentration in the range of 5 mM to 50 mM. 0 mM histidine, or a final concentration of 1 mM to 10 mM succinate. The final concentration of histidine was 20 mM±2 mM.

[0147] Saline (i.e., a solution containing NaCl) is preferred, but other suitable formulations are also suitable. Salts include CaCl2, KCl, and MgCl2, and combinations thereof but are not limited to sucrose, trehalose, mannitol, sorbitol and Substituting non-ionic tonicity agents, including but not limited to glycerol, for salt Suitable salt ranges include 25 mM to 500 mM or 40 mM to 170 mM. In one embodiment, the saline solution contains, but is not limited to, NaCl. l 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 , comprising one or more pneumococcal polysaccharides conjugated to a carrier protein. Quality is CRM 197 , diphtheria toxin fragment B (DTFB), DTFBC8, diphtheria Tetanus toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, Latoxoid, E. coli LT, E. coli ST, Pseudomonas aeruginosa exotoxin A, and combinations thereof In one embodiment, all polysaccharide-protein conjugates can be selected from the following: The gates are prepared using aqueous chemistry. The above polysaccharide-protein conjugates are prepared using DMSO as a solvent. , polysaccharide-protein conjugate formulations are effective against serotypes 6A, 6B, 7F, 18C, and 19A Polysaccharide-protein conjugates derived from 19F and 23F were synthesized using DMSO solvent. Polysaccharide proteins derived from serotypes 1, 3, 4, 5, 9V, 14, 22F, and 33F were prepared. The 15-valent pneumococcal conjugate (1 5vPnC) formulation.

[0150] In another embodiment, the pharmaceutical composition is delivered in a sustained release system. For example, the agent is administered intravenously. The compound may be administered using an injection, a transdermal patch, liposomes, or other modes of administration. In embodiments, the polymeric material is used, for example, in microspheres or implants. will be done.

[0151] The compositions of the present invention may also comprise one or more proteins derived from Streptococcus pneumoniae. Examples of Streptococcus pneumoniae proteins suitable for inclusion include those described in WO 02 / 0838 55 and 02 / 053761.

[0152] Although various embodiments of the present invention have been described with reference to the accompanying description and drawings, the present invention is not limited thereto. It is understood that the present invention is not limited to these precise embodiments, but rather encompasses the scope of the invention as defined in the appended claims. Various changes and modifications may be made by those skilled in the art without departing from the scope or spirit of the present invention. I want you to understand that.

[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 Immunochemist See, s. ry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, e.g., European Patent No. 0497524. Pneumococcal subtype isolates include: These cells are available from the American Type Culture Collection (Manassas, Virginia). The organism is a encapsulated, non-motile, Gram-positive, lancet-shaped diplococci (blood The subtypes are identified as Q-hemolytic (α-hemolytic on agar). They can be identified based on the uelling reaction. For example, U.S. Patent No. 5,847, See issue 112.

[0155] Cell banks representing each of the S. pneumoniae serotypes present were prepared using Merck Culture Collection (C). The product was obtained in frozen vials from the Collection (Rahway, NJ). The thawed seed culture is transferred to a seed fermenter containing pre-sterilized growth media appropriate for S. pneumoniae. The culture was grown in a seed fermentor under temperature and pH control. The total volume of the seed fermentor was The culture was transferred to a production fermenter containing pre-sterilized growth media. During the cell growth stage, the temperature, pH and agitation speed were controlled.

[0156] The fermentation process was terminated by the addition of an inactivating agent. After inactivation, the batch was transferred to an inactivation tank. The cells were transferred to a centrifuge and kept under controlled temperature and agitation. The batch was then subjected to ultrafiltration and diafiltration. The batch was then subjected to solvent separation. This was separated to remove impurities and recover the polysaccharide.

[0157] Example 2: Serotypes 1, 3, 4, 5, 6A, 6B, 7 using reductive amination in aqueous solution CRMs for F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F 197 Conjugation to Different polysaccharide serotypes were purified using a common process flow. 197 Carrier Tamper Polysaccharides are dissolved, reduced in size, and chemically activated. The purified CRM is then purified by ultrafiltration. 197 the reaction mixture NiCl2 (2 mM) in the solution was used to conjugate the activated polysaccharide, and the resulting conjugate The dugate was purified by ultrafiltration followed by a final filtration through a 0.2 micron filter. Some process parameters within each step, such as pH, temperature, concentration and time, are In the chapter, it is controlled by serotype-specific values.

[0158] Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and all serotypes except serotype 19A were diluted to 0. All serotypes except serotype 19A were homogenized and filtered through a 45 micron filter. Serotype 19A has a relatively small starting size, so the size The homogenization pressure and homogenization were adjusted to achieve serotype-specific molecular mass. The number of passes through the isotherm was adjusted to the serotype-specific target value (150-1000 bar; 4-7 passes). The size-reduced polysaccharide was filtered through a 0.2 micron filter and then concentrated. and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

[0159] The polysaccharide solution was then incubated in sodium acetate buffer at serotype-specific temperatures (4–22°C) and The pH was adjusted to 4-5 to minimize the size reduction of polysaccharides due to activation. For serotypes (except serotype 4), 100 mM sodium metaperiodate solution was added. Polysaccharide activation was initiated. The amount of sodium metaperiodate added was serotype-specific. In the range of about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of sodium metaperiodate was shown to achieve the desired level of polysaccharide activation. (moles of aldehyde per mole of polysaccharide repeating unit). For this purpose, the batch is heated to approximately 50°C and pH 4.1 before adding the sodium metaperiodate. Incubation resulted in partial deketalization of the polysaccharide.

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

[0161] CRM197 Conjugation of polysaccharides to The oxidized polysaccharide solution was prepared, depending on the serotype, in a mixture of water and 1.5 M potassium phosphate (pH 6.0 or pH 7.0). The pH of the selected buffer is important for the conjugation reaction. The purpose of this study was to improve the stability of activated polysaccharides in aqueous solutions. 12 / 173876), and the purified product obtained by expression in Pseudomonas fluorescens CRM 197 Filter through a 0.2 micron filter and 0.4 to 1.0 wt. depending on the serotype. / w range of polysaccharides vs. CRM 197 The resulting conjugates were combined with a buffered polysaccharide solution in a weight ratio of 1:1. Polysaccharides vs. CRM in ducate 197 The mass ratio was chosen to control the ratio. The phosphate concentration is serotype specific and ranges from 3.6 to 10.0 g / L, respectively, depending on the serotype. The L and β ranged from 100 to 150 mM. The serotype-specific polysaccharide concentrations were selected to obtain the desired results. The solution was then filtered through a 0.2 micron filter. Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution. Sodium borohydride (2 moles per mole of polysaccharide repeating unit) was added. The incubation was allowed to proceed for a serotype-specific period (72-120 hours) to determine the polysaccharide and protein content. Consumption was maximized.

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

[0163] Final Filtration and Product Storage The batch was then concentrated and filtered at 4 °C using a 300 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltration against 10 mM L-histidine in 150 mM sodium chloride (pH 7.0) 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, and the 100 kDa N Using a MWCO tangential flow ultrafiltration membrane, filter the solution in 150 mM sodium chloride (pH 7.0) at 4 °C. Diafiltered against 10 mM L-histidine and filtered through a 0.2 micron filter. .

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

[0166] Example 3: Serotypes 3, 4, 6A, and 6 using reductive amination in dimethyl sulfoxide CRMs for B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F 197 Conjugation Methods Different polysaccharide serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F are purified using a typical process flow. 197 Responsible The polysaccharides are dissolved and saturates to the desired molecular mass. The activated polysaccharides are then lysed, chemically activated, and buffer exchanged by ultrafiltration. and purified CRM 197 were individually lyophilized and redissolved in dimethyl sulfoxide (DMSO). The reconstituted polysaccharide solution and CRM were then 197 Combine the solutions as follows: The resulting conjugate was purified by ultrafiltration and then The final filtration was carried out using a 0.2 micron filter. Some process parameters within the process are controlled to serotype-specific values ​​in the following chapters. .

[0167] Polysaccharide size reduction and oxidation Purified pneumococcal capsule Ps powder was dissolved in water, and all serotypes except serotype 19A were diluted to 0. All serotypes except serotypes 18C and 19A were homogenized. The molecular mass of Ps was reduced by homogenization. The blood pressure was controlled to a serotype-specific target value (150-1000 bar; 4-7 passes). The purified form 18C was reduced in size by acid hydrolysis at temperatures above 90°C.

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

[0169] The polysaccharide solution was then incubated in sodium acetate buffer at serotype-specific temperatures (4–22°C) and The pH was adjusted to 4-5 to minimize the size reduction of polysaccharides due to activation. For serotypes (except serotype 4), 100 mM sodium metaperiodate solution was added. Polysaccharide activation was initiated. The amount of sodium metaperiodate added was serotype-specific. In the range of about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of sodium metaperiodate was shown to achieve the desired level of polysaccharide activation. (moles of aldehyde per mole of polysaccharide repeating unit). For this purpose, the batch is heated to approximately 50°C and pH 4.1 before adding the sodium metaperiodate. Incubation resulted in partial deketalization of the polysaccharide.

[0170] The activated product was filtered through a 10 kDa NMWCO tangential flow ultrafiltration membrane in 10 mM phosphate buffer. Diafiltration against potassium (pH 6.4) followed by diafiltration using a 10 kDa NMWCO membrane The solution was diafiltered or dialyzed against water. 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 As previously described (WO 2012 / 173876), Pseudomonas Purified CRM obtained through expression in C. fluorescens 197 , 5 kDa NMWCO Diafiltration was performed against 2-5 mM phosphate (pH 7.0) buffer using a tangential flow ultrafiltration membrane. The mixture was filtered through a 0.2 micron filter.

[0172] For serotypes other than serotype 3, oxidized polysaccharides were added in water at 6 mg Ps / mL and The serotype 3 was formulated with 5% w / v sucrose (50 mg sucrose / mL). in water at 2 mg Ps / mL and 10% w / v sucrose (100 mg sucrose / mL The protein solution was formulated at 6 mg Pr / mL and and 1% w / v sucrose (10 mg sucrose / mL).

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

[0174] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 per mole of polysaccharide repeating unit) was added. Approximately 0.025% (w / v) polysorbate 20 was added at approximately 4°C. The batch was diluted in 150 mM sodium chloride with or without potassium phosphate. Buffer was added to neutralize the pH. Serotypes 3, 6A, 6B, 7F, 9V, 18C, and 19 For A, 19F, 22F, 23F and 33F, the batch was concentrated and 30 kDa N MWCO tangential flow ultrafiltration membranes were used to filter the solution in 25 mM potassium phosphate (pH 7) at approximately 4°C. The solution was diafiltered against 150 mM sodium chloride with or without HCl.

[0175] Final Filtration and Product Storage Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 22F, 23F, and 33F Concentrate and filter to a concentration of 0.015% ( 150 mM sodium chloride (pH 7.5) with or without (w / v) polysorbate 20 The retentate batch was diafiltered against 10 mM histidine in 0.2 micron filter paper. The mixture was filtered through a filter.

[0176] Serotype 19F was incubated for approximately 5 days and then subjected to 300 kDa NMWCO tangential flow ultrafiltration. The membrane was used to prepare a solution of 10 mM histidine in 150 mM sodium chloride (pH 7.0) at approximately 4°C. The solution was diafiltered against water and filtered through a 0.2 micron filter.

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

[0178] Serotypes 4 and 14 were incubated at 150 mM MgCl using a 300 kDa NMWCO membrane at approximately 4°C. Dialyze against sodium chloride, filter through a 0.2 micron filter, and dispense into aliquots and frozen at ≦−60°C.

[0179] Example 4: Analysis of the conjugates Conjugate molecular weight and concentration using HPSEC / UV / MALS / RI assay degree analysis Inject the conjugate sample and subject it to high performance size exclusion chromatography (HPSEC). Detection was by ultraviolet (UV), multi-angle light scattering (MALS), and refractive index in series. Protein concentration was determined using the UV280 extinction coefficient. The change in refractive index of the solution and the change in solute concentration (reported in mL / g) are expressed as dn / d The c coefficient was used to calculate the polysaccharide concentration from the RI signal (contributed by both protein and polysaccharide). The average molecular weight of the sample was determined by the measured concentration and optical density across the entire sample peak. The scattering information was used to analyze the data using Astra software (Wyatt Technology Corporation) Calculations were performed by the National Institute of Standards and Technology, Santa Barbara, CA.

[0180] Polysaccharide activation assay Conjugation occurs via the binding of activated aldehydes to lysine residues, primarily on carrier proteins. It is produced by reductive amination and is expressed as moles of aldehyde per mole of polysaccharide repeating unit. The level of activation achieved is important for controlling the conjugation reaction. Assays for measuring .alpha.-hydroxybenzoates are described in U.S. Patent Publication No. 2017 / 0021006. There are.

[0181] Aldehyde groups (generated during periodate oxidation of polysaccharides) and thiosemicarbazide (commercial We developed an internal assay to measure activation based on reaction with ATP (available from commercial sources). It was emitted.

[0182] Quantification is by NMR (nuclear magnetic resonance) or by comparing the derivatized polysaccharides to appropriate reference standards. This is achieved by comparing the extinction coefficient of the derivative with that of the The use of extinction coefficients in this assay is comparable to the HPSEC / UV / MALS / RI method. It is similar to the use in

[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 should be run to completion, i.e. (This continues until a plateau is reached over time.) Temperature: 15 to 37°C, preferably around 21 to 27°C TSC concentration: 1~5mg / mL Reaction pH: pH 3 to 5.5, preferably 4.0 For Example 4, the polysaccharide was diluted with 1.25 to 2.5 mg / mL of thiosemicarbazide (TS C) at pH 4.0 to introduce the chromophore (for serotypes 1, 5, and 9V). (The derivatization of activated polysaccharides was performed using 1.25 mg / mL TSC). The actual time varied depending on the reaction kinetics of each serotype. TSC-Ps were isolated from TSC and other low-molecular-weight proteins by high-performance size-exclusion chromatography. The signal was detected by UV absorbance at 266 nm. Aldehyde levels were measured against a standard curve injection of Mono-TSC or by a predetermined absorbance coefficient. Mono-TSC is a synthetic thiosemicarbazone derivative of a monosaccharide. The Ps concentration measured by HPSEC / UV / MALS / RI assay was then The aldehyde level was calculated as the number of moles of aldehyde per mole of repeating unit (Ald / R Convert to U.

[0185] Thiosemicarbazide structural analogs, hydrazides, Hydrazine, semicarbazide, semicarbazide structural analogues, aminooxy compounds or aromatic Similar derivatization can be performed with aromatic amines. UV absorbance indicates the amount of amine bound to the derivatizing agent. The chromophores are either conjugated or, as in the case of thiosemicarbazide, are the result of aldehyde derivatization. It may be from a chromophore produced by

[0186] The linkage in the conjugated protein as a measure of the number of covalent bonds between the polysaccharide and the carrier protein Determining syn consumption Waters AccQ-Tag Amino Acid Analysis (AAA) was used to analyze the conjugates. The degree of conjugation in the samples was measured. To digest the samples, they were dehydrated using vapor phase acid hydrolysis in an Eldex workstation. The free amino acids were dissolved in 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized sample was then purified by chromatography on a C18 column. Analysis was performed using UPLC with UV detection. The average protein concentration was calculated based on the representative values ​​except for lysine. The lysine consumption during conjugation (i.e., lysine loss) was obtained using typical amino acids. (loss) is calculated as the difference between the average measured lysine amount in the conjugate and the expected amount of lysine in the starting protein. It was decided by.

[0187] Conjugates prepared using reductive amination in aqueous and DMSO solutions attribute Polysaccharide activity for conjugates prepared using the processes described in Examples 2 and 3 The results of activation and lysine consumption (i.e., lysine loss) are listed in Table 1. The conjugate prepared in Example 3 was similar to the conjugate prepared in aqueous solution (Example 4). 2) has a clear difference in that it has a higher lysine consumption and a lower polysaccharide activation. This is because the conjugate is prepared in DMSO solution, allowing the conjugate to be attached to the native polysaccharide structure. The polysaccharides are more readily conjugated to the carrier protein with less activation or destruction of the This suggests that the DM2 molecule binds to the ion site more efficiently than the aqueous solution. Due to the higher cross-linking in the conjugate prepared in SO solution, Gates contain, on average, more glycopeptides per polysaccharide repeat unit. The peptides are believed to be the antigenic domains against which an immune response is generated. Conjugates produced in SO4 were more immunogenic than those produced in aqueous solution. It is expected to be highly virulent.

[0188] The average molecular weight (Mw) of the conjugates in Table 1 was determined by HPSEC UV-MALS-RI. The conjugates formed by reductive amination in aqueous solution were measured by the assay. The gate was in the range of 990–3410 kDa. The proteins 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 polysaccharide binds to the N-terminal amine group of the carrier protein, or CRM 197 39 lysines in It can be conjugated to any of the side chains of the residue. 197 Amino acid composition The columns are provided in Table 2, in which lysines (abbreviated as K) are underlined. CRM 197 The degree of polysaccharide conjugation at different sites on the protein To identify and quantify the concentrations, an LC / UV / MS peptide mapping method was used. Typical conjugate samples (prepared in DMSO or aqueous solution) were double digested with trypsin. The mixture was then purified by reversed-phase C 18 Separated on a column and UV and analyzed by mass spectrometry. 197 Protein sample (conjugated with polysaccharides) The control (without trypsin) was also treated in triplicate. Since the cleavage site is on the C-terminal side of the protein, conjugation at a lysine residue is The degree of conjugation at a particular site is determined by the CRM 19 7. Determined by calculating the decrease in peak intensity of tryptic peptides compared to the control Depending on the cleavage site and sequence, the signal decrease of a particular peptide may be due to the presence of a preceding peptide. Incorrect cleavage of lysine at the end of a peptide, or incorrect cleavage of lysine at the end of a peptide, This may be due to conjugation in the middle of the amino acid sequence.

[0190] CRM 197 Peptide signals for serotype 19A conjugates compared to controls The relative percentage of reduction is plotted against the possible conjugation sites in Figure 1. The lysine positions listed on the x-axis are the CRM 197 Based on their order in the protein sequence The numbers are based on the amino acid sequence and represent the possible conjugation sites of the analyzed peptide. "33" indicates that the decrease in peptide signal is due to conjugation at the 33rd lysine. "6,7" means that the decrease in the peptide signal was due to the sixth Conjugation at the 7th or 8th lysine, or both, occurs The data in Figure 1 show that the concentration of DMSO in water was significantly higher than that in water. For the prepared conjugates, the degree of conjugation at each site was generally These results suggest that not only is the concentration of DMSO significantly higher, but also that there are many conjugation sites in DMSO. Additional conjugation sites include lysines 29, 30, 31, and 32. These consist only of lysines found in previously identified common human T cell peptide epitopes. (Raju et al., 1995, Eur. J. Immunol. 25:32 See 07-3214; CRM 197 Sequences of peptides 411-430 and peptide 43 1–450). Similar results were observed for the other serotypes tested. [Table 2]

[0191] Example 5: Serotype 3Ps-CRM prepared in aqueous solution 197 Conjugates and DMS Serotype 3Ps-CRM prepared in O 197 Mouse immunogenicity compared to conjugates test All animal experiments followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was strictly adhered to at the MRL in West Point, Pennsylvania. Approved by the Institutional Animal Care and Use Committee (IACUC).

[0192] Eight-week-old female CD1 mice were purchased from the MRL Animal Research Laboratory in West Point, PA. They were housed in microisolator cages (n=10 / cage) at the facility. Food and water were available ad libitum. Mice (n=10 / group) were treated with aluminum phosphate as described in Table 3. ST3-CRM formulated with anti-inflammatory drug (APA) 197 Conjugate (0 Negative control animals received APA alone. Immunizations were given on days 0, 14, and 28. On days 6 and 34, blood was collected. The fluid was then injected via the tail vein into a serum separator tube (BD, Franklin Lakes, NJ). ) was collected. [Table 3]

[0193] Electrochemiluminescence (ECL) immunogenicity assay Mouse antibody responses were only slightly higher in a 96-well multiplex electrochemiluminescence assay. Measurements were performed as previously described with minor modifications. Marchese et al. 2009,Clin Vaccine Immunol 16(3):387-96;S Kinner et al., 2011, Vaccine 29(48):8870-6 and Caro-Aguilar et al., 2017 Vaccine 35(6 ):865-72. Briefly, see Meso-Scale Discovery Plates (Meso Scale Diagnostics, Rockville, MD) ) for 1 hour, followed by washing and then 25 μl of 2 μg / ml Sulfo-tag (Meso Scale Diagnostics, Maryland) A 100-well plate containing goat anti-mouse IgG (Rockville, TX) was added to each well. Incubate at room temperature for 1 hour, then process as described above and add MESO Sector S It was read at 600.

[0194] The cut-off value (the geometric mean signal of pneumococcal polysaccharide ECL of a given positive control pooled mouse serum) ECL titers were calculated as the reciprocal of the linearly interpolated dilution corresponding to the ECL logarithmic scale. and dilution were used for interpolation. The linearly interpolated dilution was then back-transformed to obtain Titers were obtained from the last three ECL data for sample curves that were completely above the cutoff line. For sample curves using the intercept and slope of the data points or completely below the cut-off line Linear extrapolation (log-log scale) using the intercept and slope of the first two ECL data points Based on this, the titer was determined for samples outside the test dilution range of 100 to 1,562,500. Titers were then obtained by back-transforming the linearly extrapolated dilutions.

[0195] Opsonophagocytic killing assay (OPA) The opsonophagocytic killing assay (OPA) for pneumococcal serotype 3 was performed with minor modifications. and was performed as previously described (Caro-Aguilar et al., 201 7 Vaccine 35(6):865-72 and Burton et al.,2 006, Clin Vaccine Immunol 13(9):1004-9). blood After incubation of supernatant, bacteria, complement, and HL-60 cells, 10 μl of opsonized phagocytic The working reaction was transferred to a 96-well Millipore tube containing 200 μl / well of sterile water. The plate was vacuum filtered and 100 μl of T Odd Hewett yeast extract (THYE, Teknova) broth was added. The solution was filtered and the wet plates were placed in a sealed plastic bag overnight at 27°C. Then, plate filters were filled with 100 μl / well of 0.1% Coomassie Blue solution (Bio-R The stain was filtered through the plate. The colonies were decolorized with Coomassie decolorizing solution (Bio-Rad) and vacuum filtered again until dry. The stained bacterial colonies were analyzed using a CTL Immunospot reader (Shenyang, OH). OPK titers were calculated using the average of the complement control (serum-free control) wells. The serologic response was defined as the reciprocal serum dilution with at least 50% killing compared to proliferation. The signal was linearly interpolated between successive dilutions that bracketed 50% kill. So I calculated it.

[0196] The results before and after the third dose are shown in Figure 2 and Table 4 for ECL immunogenicity. For OPA, see Figure 3. By using the process with water and DMSO solutions, Both conjugates prepared were immunogenic and provided functional killing activity against bacteria. Interestingly, the conjugates prepared by the process using DMSO solution The conjugates exhibited higher ECL immunogenicity and OPA responses than conjugates prepared using aqueous solutions. The difference in ECL immunogenicity was statistically significant. The MT ratio is 3.41 (the lower and upper limits of the 95% confidence interval are 1.26 and 1.41, respectively). and 9.26). [Table 4]

[0197] Example 6: Pneumococcal polysaccharide-protein conjugates prepared using reductive amination in aqueous solution Conjugate and pneumococcal polysaccharide-protein complexes prepared using reductive amination in DMSO. Immunogenicity study in adults compared with Jugate In this example, two 15-valent pneumococcal vaccines were administered to healthy, unvaccinated adults aged 50 years or older. To describe the immunogenicity and safety of the pneumococcal conjugate vaccine (PCV15). do.

[0198] Test Design Healthy adult subjects aged 50 years or older (if they have underlying chronic diseases, they must be in a stable state) Two different PCV15 formulations (PCV15- PCV15-A and PCV15-B) and Prevnar 13™ (pneumococcal 13-valent conjugate) Diphtheria CRM vaccine 197 Protein], a subsidiary of Pfizer Inc. Yeth Pharmaceuticals (Philadelphia, Pennsylvania, USA) A randomized, multicenter, double-blind study was conducted to compare the safety, tolerability, and immunogenicity of a single dose of The blinded study was conducted in accordance with Good Clinical Practices.

[0199] A total of 690 healthy, unvaccinated individuals aged 50 years or older were enrolled in three different trials. Vaccination groups: Prevnar 13™, PCV15-A and PCV15-B Randomization was based on age at study entry (50-64 years, 6 Patients were stratified by age (5–74 years, and ≥75 years).

[0200] PCV15 is CRM 197 The following 2µg / 0.5mL doses of Each serotype (1,3,4,5,6A, 7F, 9V, 14,18C, 19A, 19F, 22F , 23F, 33F) pneumococcal polysaccharides, CRM 197 Conjugated to 4μg / 0. 5 mL dose of serotype 6B pneumococcal polysaccharide, 125 μg / 0.5 mL dose of aluminum phosphate adjuvant, 20 mM L-histidine, 150 mM sodium chloride (pH 5.8 PCV15-A was formulated with 0.2% w / v P188. V15-B was formulated with 0.1% w / v PS-20.

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

[0202] PCV15-B, serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F, CRM was synthesized using reductive amination in DMSO as described in Example 3. 197 Conjugated to The properties of these conjugates (materials of conjugate lot no. 2) are listed in Table 1. The remaining serotypes (1, 3, 4, 5, 9V, 14, 22F, and 33F) The conjugate is the same conjugate used in PCV15-A.

[0203] Both PCV15 formulations were generally P based on cumulative safety assessments (data not shown). It had a safety profile comparable to that of Revnar 13™. The GMC of gG and the GMT of OPA were measured on day 30 (OPA results are not included). do not have).

[0204] result IgG geometric mean concentrations (GMC) and confidence intervals (CI) are summarized in Table 6. PCV15 -A and serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F conjugates were prepared using different conjugation processes as described above. Consistent with the results shown in Table 4, the immunogenic responses for each serotype are shown in Table 6. The polysaccharide serotype was found to be CRM in DMSO. 197 This was improved when conjugated to The GMCs of serotypes 18C, 19A, 19F, and 23F in PCV15-B were These data suggest that the immunogenicity of IFN-γ was significantly higher than that of IFN-γ in IFN-γ-A (two-sided α = 0.05). This finding strongly demonstrates the advantage of conjugation in DMSO to improve has not been demonstrated for pneumococcal or other conjugate vaccines. not present. [Table 5]

Claims

[Claim 1] Pneumococcal serotype 3 polysaccharide-CRM by reductive amination １９７ 1. A method for preparing a carrier protein conjugate, comprising: a) reacting pneumococcal serotype 3 polysaccharide with an amount of an oxidizing agent to form activated pneumococcal serotype 3 polysaccharide; and b) reacting the activated pneumococcal serotype 3 polysaccharide with CRM in an aprotic solvent; １９７ React with a carrier protein to form pneumococcal serotype 3 polysaccharide-CRM １９７ Forming a carrier protein conjugate Including, The obtained pneumococcal serotype 3 polysaccharide-CRM １９７ The carrier protein conjugate is a carrier protein conjugate according to steps a) and b), except that the activated pneumococcal serotype 3 polysaccharide is combined with a CRM in an aqueous solution. １９７ Pneumococcal serotype 3 polysaccharide-CRM prepared by reacting with a carrier protein １９７ The method, wherein the antibody has higher antigenicity as measured by opsonophagocytic killing assay (OPA) and electrochemiluminescence (ECL) immunogenicity assay compared to a carrier protein conjugate, and the aprotic solvent is dimethyl sulfoxide (DMSO).

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