Methods for formulating pneumococcal polysaccharides for conjugation to carrier proteins - Patent Application 20070122997

Abstract

To provide a manufacturing condition related to conjugation of capsular polysaccharide from Streptococcus pneumoniae to carrier protein.SOLUTION: A method for producing a polysaccharide-protein conjugate includes allowing polysaccharide in first solution to react with protein in second solution to from third solution, and performing polysaccharide-protein conjugation reaction in the third solution to create a polysaccharide-protein conjugate, where the third solution contains at least 1 mM of salt. It is preferable that the salt be sodium salt, potassium salt, lithium salt, magnesium salt or calcium salt. A polyvalent pneumococcal vaccine can contain the polysaccharide-protein conjugate produced by using a process of the present invention.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for the production of Streptococcus pneumoniae. Many process improvements related to the conjugation of capsular polysaccharides to carrier proteins The polysaccharide-protein conjugates produced using the methods of the present invention are multivalent. It can be included in pneumococcal vaccines. [Background technology]

[0002] Streptococcus pneumoniae is an encapsulated bacterium. It is one of the most important causes of serious illness worldwide. Centers for Disease Control and Prev The Centers for Disease Control and Prevention (CDC) estimates that there are 3,000 cases of pneumococcal meningitis and pulmonary meningitis annually in the United States. 50,000 cases of Streptococcus pneumoniae bacteremia, 7,000,000 cases of pneumococcal otitis media, and The Centers for Disease Control and Prevention estimates that there were 500,000 cases of pulmonary emphysema. se Control and Prevention, MMWR Morb Mor See tal Wkly Rep 1997, 46(RR-8):1-13. Furthermore, the complications of these diseases can be severe, and several studies have shown that pneumococcal myelopathy Up to 8% mortality and 25% neurological sequelae have been reported for meningitis. See t al., 1998, Pediatrics 102:1087-97 . [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46(RR-8):1-13 [Non-patent document 2] Arditi et al., 1998, Pediatrics 102:1087-97 Summary of the Invention [Problem to be solved by the invention]

[0004] The multivalent pneumococcal polysaccharide vaccine, which has been licensed for many years, is used in adults, especially the elderly and It has proven to be highly beneficial in preventing pneumococcal disease in high-risk individuals. However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides. Bacterial polysaccharides induce weak or no responses in infants, a T cell-independent mechanism The chemical conjugation of bacterial polysaccharide immunogens to carrier proteins is Alters immune responses to T-cell-dependent immunogens in infants. Diphtheria toxoid (DTx, a chemically detoxified version of DT) and CRM197 have the T It has been reported to be a carrier protein for bacterial polysaccharide immunogens due to the presence of cell-stimulating epitopes. It is being done.

[0005] At the time, the pneumococcal vaccine, Prevnar®, was used in infants and toddlers. The seven most frequently isolated serotypes causing invasive pneumococcal disease ( 4 , 6 B, 9V, 14, 18C, 19F and 23F) and was approved in the United States in February 2000. It was first approved in the United States after Prevnar® became widely available. reported that the serotypes present in Prevnar® may increase the risk of invasive pneumococcal disease in children. There has been a significant decrease in cases. and Prevention, MMWR Morb Mortal Wkly Re See p 2005, 54(36):893-7. However, in certain parts of the world In the US, there are limitations to the serotype coverage provided by Prevnar®. There is some evidence of new serotypes (e.g., 19A) of et al., 2004, Am J Epidemiol 159:634-44 ;Whitney et al., 2003,N Engl J Med 348:1 737-46;Kyaw et al., 2006, N Engl J Med 35 4:1455-63;Hicks et al., 2007, J Infect Di. s 196:1346-54;Traore et al., 2009,Clin I Infect Dis 48:S181-S189.

[0006] Prevnar 13® is a serotype 1, 3, 4, 5, 6A, 6B, 7F, and 9V 13-valent pneumococcal polysaccharide-protein complex containing 14, 18C, 19A, 19F, and 23F Adjugate vaccines. See, for example, U.S. Patent Application Publication No. US2006 / 02283 80A1, Prymula et al, 2006, Lancet 367:740 -48 and Kieninger et al, Safety and Immunol ogic Non-inferiority of 13-valent Pneumo coccal Conjugate Vaccine Compared to 7-v alent Pneumococcal Conjugate Vaccine Giv en as a 4-Dose Series in Healthy Infants and Toddlers (the 48 th Annual ICAAC / ISDA 46 th Annual Meeting, Washington DC, Oct. (published in Ober 25-28, 2008). Dagan et al., 1 998, Infect Immun. 66: 2093-2098 and Fattom , 1999, Vaccine 17: 126.

[0007] Current multivalent pneumococcal vaccines have been shown to reduce the risk of pneumococcal disease associated with the serotypes present in the vaccine. However, the vaccine was effective in reducing the incidence of pneumonia. The prevalence of bacterially expressed serotypes is increasing. The efficacy of each serotype and the unique challenges that certain serotypes present will be determined. Therefore, novel pneumococcal serotypes should be conjugated for inclusion in future vaccines. Improved gating process conditions are needed. [Means for solving the problem]

[0008] The present invention provides a method for the detection of Streptococcus pneumoniae (Streptococcus pneumoniae) that is specific for a particular serotype. A number of process modifications in the production of polysaccharides (Ps) from Pseudomonas neumoniae are presented.

[0009] These process modifications improve the properties of the polysaccharide and / or polysaccharide dissolution, thereby enhancing the conjugate This will improve the gaming experience.

[0010] In one embodiment, the present invention provides a method for preparing a carrier protein (Pr) in an aprotic solvent. Size-reduced serotype 12 that exhibits desirable conjugation properties when conjugated S. pneumoniae polysaccharides from F, 23A, 24F and 31 were obtained. Provides process conditions for

[0011] Specifically, the reduction in Ps size of these serotypes by acid hydrolysis resulted in a significantly greater reduction in Ps size compared to homogenization. This results in a lower molecular weight of Ps for protein conjugation, thereby reducing lysine consumption. , free Ps or free Pr.

[0012] In one embodiment, the present invention relates to serotypes 15A, 16F, 17F, 20, 24F, and 35B, sodium chloride was used for the S. pneumoniae polysaccharides. Improved after conjugation in aprotic solvents such as DMSO with methyl methylpropional The process conditions for obtaining the desired polysaccharide-protein conjugate properties are provided. In this embodiment, ≧1 mM salt is added before or during the conjugation reaction. By including sodium chloride (at what stage in the process do you add the sodium chloride? (irrespective of the presence of phospholipids), increased conjugate size, increased lysine consumption, free P or free P This results in improved conjugate properties such as reduced Pr dissociation.

[0013] In one embodiment, the present invention provides a method for preparing serotype 3, 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, 39, 38, Various pre-lyophilized formulations of 8 and 24F Streptococcus pneumoniae polysaccharides Specifically, the polysaccharide is prepared in a mass ratio of sucrose to polysaccharide of ≥ 30, and optimally is formulated with sucrose and water at a concentration of ≥ 40 times. For example, a given pre-lyophilized For a polysaccharide concentration of 2 mgPs / mL, the sucrose concentration should be at least 6 mgPs / mL for dissolution after lyophilization. 0 mg sucrose / mL (6% wt / vol sucrose), optimally ≥ 80 mg sucrose / mL The concentration should be 1000 mg / mL (8% wt / vol sucrose).

[0014] Polysaccharides formulated in these ways allow for the formation of complexes with proteins after lyophilization and reconstitution. This allows for jugation to achieve the desired properties. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the effect of sodium chloride on conjugate size for S. pneumoniae polysaccharides from serotype 20. [Figure 2] FIG. 1 shows the effect of sodium chloride on lysine consumption for S. pneumoniae polysaccharides from serotype 20. [Figure 3] FIG. 1 shows the effect of sodium chloride on free Ps and Pr for S. pneumoniae polysaccharides from serotype 20. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a method for the detection of Streptococcus pneumoniae (Streptococcus pneumoniae) that is specific for a particular serotype. A number of process modifications in the production of polysaccharides (Ps) from Pseudomonas neumoniae are presented. These process modifications improve the properties of the polysaccharide and / or polysaccharide dissolution, thereby enhancing the conjugate This will improve communication.

[0017] The inventors have demonstrated that certain pneumococcal chains can be synthesized by acid hydrolysis prior to protein conjugation. As shown in the examples, the size reduction of S. pneumoniae polysaccharides We have found that this results in improved conjugate properties. The use of acid hydrolysis is not a reliable method, but may explain the observed behavior. One possible mechanism for this is conjugation with low molecular weight Ps from acid hydrolysis. The steric hindrance between Ps and Pr molecules during the conjugation reaction is small, and Thus, the interaction may be promoted and conjugation may be improved.

[0018] The present inventors have demonstrated that certain S. pneumoniae polysaccharides can be completely dissolved. As shown in the examples, we discovered that the presence of sucrose after freeze-drying is necessary to achieve this. Without being bound by any particular theory, we have found that the observed dissolution behavior can be explained by One possible mechanism for the possible use of sucrose is that some polysaccharide serotypes However, due to its chemical structure, it self-associates more readily than other serotypes upon lyophilization or thawing, resulting in synaptic The relatively high sugar / polysaccharide ratio inhibits self-association, allowing for dissolution after lyophilization. This is what it means.

[0019] As shown in the Examples, the present inventors have found that the conjugation reaction In particular, the inclusion of ≥ 1 mM sodium chloride in the serotypes 16F, 20, and 24F Regarding pneumococcal polysaccharides (at what stage in the process do you add sodium chloride? Regardless of whether or not the conjugate size is increased, lysine consumption increases, free Ps or free P It has been found that this results in improved conjugate properties such as a decrease in r. Without being bound by theory, the conjugation observed with sodium chloride One possible mechanism for the use of sodium chloride that may explain the reaction behavior is The mechanism is that for some polysaccharide serotypes, due to their chemical structure and charge distribution, sodium chloride The electrostatic shielding provided by the ammonium phosphate facilitates the interaction with the protein, resulting in conjugation. Sodium chloride provides additional solubility to the reaction mixture. It also provides ionic strength, thereby reducing the exposure of hydrophobic regions of the carrier protein. , protein aggregation may be reduced.

[0020] As used herein, the term "polysaccharides" (Ps) refers to the polysaccharides used in immunology and bacterial vaccines. Antigenic sugar elements (or antigenic units) commonly used in the field of immunology, including but not limited to: Although there are no "sugars," "oligosaccharides," "polysaccharides," "liposaccharides," and "lipo-oligosaccharides (LOS)," ", "Lipopolysaccharide (LPS)", "Glycosylate", "Conjugate Polysaccharide", "Derivatized or Unless otherwise specified, the term "activated polysaccharides or oligosaccharides" is intended to include "activated polysaccharides or oligosaccharides" and the like. The nomenclature of polysaccharides used in the IUB-IUPAC Joint Commission on Biochemical Nomenclature(JCBM) Recom Follow the 1980 recommendations. JCBN, 1982, J. Biol. See Chem. 257:3352-3354.

[0021] As used herein, an "immunogenic composition" refers to an immunogenic composition that is humorally or cell-mediated or otherwise Both are bacterial capsular polysaccharides that have the ability to induce an immune response in a host, such as a mammal. or a composition containing an antigen such as a polysaccharide-protein conjugate. They act to sensitize the host by presenting antigens in cooperation with MHC molecules on the cell surface. Furthermore, antigen-specific T cells or antibodies may be generated to allow for future protection of the immunized host. Thus, the immunogenic composition can protect the host from infection by bacteria, It can reduce the severity or protect the host from death due to bacterial infection. The immunogenic compositions can also be used to generate polyclonal or monoclonal antibodies. These immunogenic compositions can be used to provide passive immunity to subjects. by bacterial killing in animal efficacy models or via opsonization-killing assays. Antibodies can also be generated that have the functionality to be measured.

[0022] As used herein, the term "isolated" in relation to polysaccharides means isolated by centrifugation. , depth filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration and / or from the purified polysaccharides using purification techniques known in the art, including the use of column chromatography. It refers to the isolation of serotype-specific capsular polysaccharides of Streptococcus pneumoniae (S. pneumoniae). The isolated polysaccharides are obtained by partial removal of proteins, nucleic acids, and non-specific endogenous polysaccharides (C-polysaccharides). An isolated polysaccharide is one that contains less than 10%, 8%, 6%, 4%, or 2% protein impurities and / or The isolated polysaccharide comprises less than 20% of the C-polysaccharide relative to the type-specific polysaccharide.

[0023] As used herein, the term "purified" in the context of bacterial capsular polysaccharides It refers to the purification of polysaccharides from cell lysates by means such as centrifugation, sedimentation, and ultrafiltration. In this context, purified polysaccharide refers to the removal of cellular debris and DNA.

[0024] As used herein, the term "Mw" refers to the weight average molecular weight, typically Mw is expressed in Da or kDa. Relatively large molecules have a higher molecular mass than relatively small molecules. It is considered to comprise a relatively large portion of the total mass of the polymer sample. , by techniques such as static light scattering, small angle neutron scattering, X-ray scattering and sedimentation velocity can be done.

[0025] As used herein, the term "Mn" refers to the number average molecular weight, typically It is expressed in Da or kDa. Mn is calculated by dividing the total weight of the sample by the number of molecules in the sample. Viscosity measurements via gel permeation chromatography (Marc-Houwink equation) by techniques such as vapor pressure osmometry, end group quantification or proton NMR. Mw / Mn reflects the polydispersity.

[0026] As used herein, "comprising" when used in the immunogenic compositions of the invention The term "consists of" does not include any other components (for antigen mixtures, the language restriction "consists of" is used). (subject to limitations), e.g., adjuvants and excipients. When used with respect to a conjugate mixture, the term "consisting of" refers to a specific Streptococcus pneumoniae S. pneumoniae polysaccharide-protein conjugates from different serotypes Other S. pneumoniae polysaccharide-protein conjugates This refers to a mixture that does not have a high

[0027] Unless otherwise stated, all ranges provided herein include the lower and upper limits recited. This includes the upper limit.

[0028] capsular polysaccharide Pneumococcus from the serotypes of the present invention (e.g., serotypes 23A, 24F, and 31) Capsular polysaccharides from Coccus pneumoniae are prepared using standard methods known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and are available in publicly available forms. by known methods (see, for example, European Patent Nos. EP497524 and EP497525). and by microfluidization, preferably performed using a homogenizer, or chemical Some sizing can be achieved by hydrolysis. Growth of S. pneumoniae strains corresponding to the serotype in soy-based media The individual polysaccharides are then purified by standard steps such as centrifugation, precipitation, and ultrafiltration. See, e.g., U.S. Patent Publication No. 2008 / 0286838 and U.S. Patent No. 5,847,111. See No. 2. Polysaccharides may be sized to reduce viscosity and / or improve subsequent conjugation. The filterability of the gated product can be improved. Chemical hydrolysis is carried out using acetic acid. Mechanical sizing can be performed using high pressure homogenizing shear.

[0029] For serotypes 12F, 23A, 24F, and 31, homogenization of polysaccharides from these serotypes It was found that the desired properties were not obtained with serum other than 12F. See Example 3. For the mold, the polysaccharide batch is heated to 80-92°C, preferably 90°C, and acetic acid, hydrochloric acid, Add an acid, such as phosphoric acid or citric acid, to a final concentration of 50-200 mM, then add at least Incubate for 10, 20, 30, 40, or 50 minutes. Acid hydrolysis can be performed. In certain embodiments, acid hydrolysis can be performed for up to 90 minutes. and continue for up to 150 or 155 minutes. After the incubation period is over, For example, add concentrated potassium phosphate pH 7 buffer to a final concentration of 400 mM and cool to ≤22°C. The batch is neutralized by 0.2 micron filtration and concentrated. and diafiltered against water using a 5-10 kDa NMWC tangential flow ultrafiltration membrane. .

[0030] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of between 5 kDa and 4,000 kDa. The molecular weight is measured by multi-angle light scattering (MALS) and refractive index detectors. Calculation by size exclusion chromatography (SEC) combined with (RI) In other such embodiments, the polysaccharide is between 10 kDa and 4,000 kDa; 50kDa~4,000kDa;50kDa~3,000kDa;50kDa~2,00 0kDa;50kDa~1,500kDa;50kDa~1,000kDa;50kDa ~750kDa;50kDa~500kDa;100kDa~4,000kDa;100 kDa~3,000kDa;100kDa~2,000kDa;100kDa~1,50 0kDa;100kDa~1,000kDa;100kDa~750kDa;100kDa a~500kDa;100~400kDa;200kDa~4,000kDa;200k Da~3,000kDa;200kDa~2,000kDa;200kDa~1,500 kDa; 200 kDa to 1,000 kDa; or 200 kDa to 500 kDa average molecular weight In certain embodiments, the average molecular weight is 50 to 300 kD.

[0031] In certain embodiments, the polysaccharide may have a molecular weight of 10 to 10,000, 10 to 5,000, 10 to In certain embodiments, the polysaccharide has 4,000 or 10-1000 repeating units. , 20 to 400, 30 to 300, 40 to 200, or 50 to 100 repeating units In certain embodiments, the polysaccharide has between 40 and 900 repeating units.

[0032] Carrier proteins Multiple strains from one or more of the S. pneumoniae serotypes described herein The sugars can be conjugated to carrier proteins to deliver the drug to children, the elderly, and / or immunocompromised subjects. When multiple serotypes are used in a multivalent composition, the immunogenicity in the patient can be enhanced. The serotypes may be produced with the same or different carrier proteins. Each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein. to install.

[0033] In certain embodiments of the invention, CRM197 is used as a carrier protein. CRM197 is a non-toxic mutant of diphtheria toxin (DT). The carrier protein is rendered nontoxic by a single amino acid substitution in fragment A at residue 52. In one embodiment, the CRM197 carrier protein is a mutant form of DT. Corynebacter diphtheriae grown in a casamino acid and yeast extract-based medium It is isolated from a culture of Bacillus diphtheriae strain C7 (β197). In one embodiment, CRM197 can be prepared according to the method described in U.S. Pat. No. 5,614,382. CRM197 is recombinantly obtained. Typically, CRM197 is purified by ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. CRM197 is manufactured by Pfenex Expression Technology (trade name) (Pfenex Inc., San Diego, CA) using P. fluorescens (Pseudomonas fluorescens).

[0034] Other suitable carrier proteins include DT, diphtheria toxin fragment B (DTFB), TT (tetanus toxoid) or TT fragment C, pertussis toxoid, cholera toxoid (e.g. , as described in International Patent Application Publication No. WO2004 / 083251), Escherichia coli (E. c oli)LT (heat-labile enterotoxin), E. coli (E. coli)ST (heat-stable enterotoxin) terotoxin), and from Pseudomonas aeruginosa Other inactivating bacterial toxins, such as exotoxin A. Bacterial outer membrane proteins, e.g., other Outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumococcal surface Pseudomonas aeruginosa protein A (PspA; see International Patent Application Publication No. WO 02 / 091998), pneumonia Coccal adhesion molecule protein (PsaA), a C5a peptide from group A or group B streptococci enzyme, or Haemophilus influenzae protein Protein D, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63;2706-13), for example, ply that has been detoxified in some way, e.g. dPLY-GMBS (see International Patent Application Publication No. WO04 / 081515) or dPLY - Formol, PhtX, e.g. PhtA, PhtB, PhtD, PhtE and Phttan Protein fusions, such as PhtDE fusions, PhtBE fusions (International Patent Application Publication No. W Other tamper-evident methods (see WO01 / 98334 and WO03 / 54007) can also be used. Proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum Albumin (BSA) or purified protein derivative of tuberculin (PPD), PorB ( from N. meningitidis), PD {from Haemophilus influenzae IgE (Immolophilus influenzae) protein D; e.g., European Patent EP059 4610B}, or an immunologically functional equivalent thereof, a synthetic peptide (see European Patent EP0 378881 and EP0427347), heat shock proteins (see International Patent Application See publication numbers WO93 / 17712 and WO94 / 03208), pertussis protein ( (See International Patent Application Publication No. WO98 / 58668 and European Patent EP0471177) Tokine, lymphokine, growth factor or hormone (International Patent Application Publication No. WO99 1 / 01146), multiple human CD4+ T cell epitopes from various pathogen-derived antigens Artificial proteins containing (Falugi et al., 2001, Eur J Im munol 31:3816-3824), e.g., N19 protein (Barald See oi et al, 2004, Infect Immun 72:4884-7 ), iron uptake protein (see International Patent Application Publication No. WO01 / 72337), C. diff C. difficile toxin A or B (International Patent Publication WO00 / 617 61), and flagellin (Ben-Yedidia et al., 1998, Immunol Lett 64:9) can also be used as a carrier protein. can be done.

[0035] Other DT mutants can also be used as carrier proteins, e.g., CRM1 76, CRM228, CRM45 (Uchida et al, 1973, J Bi ol Chem 218:3838-3844);CRM9, CRM45, CRM102 , CRM103 and CRM107 and Nicholls and Youle in G Enetically Engineered Toxins, Ed: Franke Other mutations described in I, Maecel Dekker Inc., 1992; deletions or Glu-148 to Asp, Gln, or Ser mutation and / or Ala158 to Gly and U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950, Other mutations disclosed in No. 740; at least one or more of residues Lys516, Lys526, Mutations of Phe530 and / or Lys534 and U.S. Pat. No. 5,917,017 or or other mutations disclosed in U.S. Pat. No. 6,455,673; or U.S. Pat. No. 5,843, This is a fragment of the disclosure in No. 711.

[0036] When using a multivalent vaccine, one or more antigens may be combined with a second carrier protein. The second carrier protein is preferably non-toxic and non-reactogenic (n It is a protein that is on-reactogenic and can be obtained in sufficient quantity and purity. The second carrier protein may also carry an antigen, such as Streptococcus pneumoniae (S. pneumoniae) niae) polysaccharides to enhance the immunogenicity of the antigen. The rear protein must be amenable to standard conjugation procedures In one embodiment, each capsular polynucleotide that is not conjugated to a first carrier protein The saccharides are conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule are conjugated to a single carrier protein. The capsular polysaccharide not conjugated to a carrier protein is conjugated to two or more carrier proteins. Conjugation to capsular proteins (each capsular polysaccharide molecule is conjugated to a single carrier protein) In such an embodiment, each capsular polysaccharide of the same serotype is typically are conjugated to the same carrier protein.

[0037] Conjugation Protein synthesis of pneumococcal polysaccharides by reductive amination in aprotic solvents such as DMSO. Conjugation to activated polysaccharides (Ps) and proteins ( Pr) is typically lyophilized and resuspended in DMSO prior to conjugation. Combine with sodium cyanoborohydride and sodium borohydride added to Details of the process are provided below.

[0038] For many pneumococcal serotypes, this process results in significant changes in size, lysine consumption, and free lysine production. Conjugates that meet the desired properties for the sugar and free protein are obtained. However, for some serotypes, the Ps and Pr concentrations and conjugation times Even after optimizing these conjugation parameters, this DMSO process Achieving the desired conjugate properties has proven relatively difficult. As described below, the present invention provides several solutions to these problems. Overcome.

[0039] Prior to conjugation, purified polysaccharides were chemically activated to allow for the transfer of the carrier protein. It is possible to prepare saccharides that have the ability to react with the saccharide to form activated polysaccharides. When used in conjunction with an activated polysaccharide, the term "activated polysaccharide" refers to a polysaccharide that has been chemically modified as described below. refers to a polysaccharide that can be conjugated to a linker or carrier protein. The purified polysaccharide may be linked to a linker. Once activated and linked to the linker, Each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. The polysaccharide conjugates of the formula (I) can be prepared by known coupling techniques.

[0040] In certain embodiments, the polysaccharide is coupled to a linker, and the free end of the linker is A polysaccharide-linker intermediate can be formed that terminates in an ester group. The carbon atom has at least one terminal end that is an ester group. The other terminal end is selected to be This allows it to react with the polysaccharide to form a polysaccharide-linker intermediate.

[0041] In certain embodiments, the polysaccharide is attached to a linker using primary amine groups in the polysaccharide. In this case, the linker typically has ester groups at both ends. This allows one of the ester groups to be converted to multiple esters by nucleophilic acyl substitution. Coupling can be achieved by reacting with the primary amine groups in the sugar. Thus, a polysaccharide-linker intermediate is formed in which the polysaccharide is coupled to the linker via an amide linkage. Thus, the linker provides the first amine group to react with the primary amine groups on the polysaccharide. and a second ester group for reacting with a primary amine group on the carrier molecule. A typical linker is N-hydroxysuccinic acid adipate. It is an acid imide diester (SIDEA).

[0042] In certain embodiments, the coupling is indirect, i.e., coupling to a linker. This can also be done with an additional linker used to derivatize the polysaccharide prior to linking. The polysaccharide is attached to the additional linker using the carbonyl group at the reducing end of the polysaccharide. This coupling can be accomplished by (a1) reacting the carbonyl group with an additional linker; and (a2) reacting the carbonyl group with an additional linker. a2) reacting the free end of the additional linker with the linker. In these embodiments, the additional linker typically has a primary amino acid at both ends. By having an amine group, one of the primary amine groups can be attached to the polysaccharide by reductive amination. Step (a1) can be carried out by reacting with the carbonyl groups in the polysaccharide. A primary amine group reactive with the hydroxyl group is used. A hydrazide group or a hydroxylamino group is preferred. Typically, the additional linker has the same primary amine group at each end. The reaction couples the polysaccharide to the additional linker via a C-N bond. A polysaccharide-additional linker intermediate is obtained.

[0043] In certain embodiments, different groups in the polysaccharide, particularly carboxyl groups, are used to differentiate the polysaccharide. can be coupled to an additional linker. This coupling is with an additional linker; and (a2) reacting the free end of the additional linker with and reacting the additional linker with a linker, wherein the additional linker is Typically, by having primary amine groups at both ends, the primary amines can be activated by EDAC. Step (a1) can be carried out by reacting one of the amino groups with a carboxyl group in the polysaccharide. It utilizes primary amine groups that are reactive with EDAC-activated carboxyl groups in polysaccharides. A hydrazide group is preferred. Typically, both ends of the additional linker have the same primary alkyl group. The reaction allows the polysaccharide to be attached to the additional linker via an amide linkage. A coupling polysaccharide-additional linker intermediate is obtained.

[0044] In one embodiment, carriers are prepared by chemical activation of polysaccharides followed by reductive amination. Conjugation to proteins is described in U.S. Pat. Nos. 4,365,170 and 4,673 ,574 and 4,902,506, U.S. Patent Application Publication No. 2006 / 0228380, 2007 / 184072, 2007 / 0231340 and 2007 / 018407 1, and International Patent Application Publication Nos. WO2006 / 110381 and WO2008 / 07965 3 and WO2008 / 143709. is an oxidizing agent that converts a primary hydroxyl group into an aldehyde, such as TEMPO( WO2104 / 097099), or by reacting two adjacent hydroxyl groups to form Oxidizing agents for aldehydes, such as periodate compounds (e.g., sodium periodate, periodic acid, This may involve activation of pneumococcal polysaccharides by reaction with a soluble ... The reaction involves random oxidation of primary hydroxyl groups or the formation of reactive aldehyde groups. Random oxidative cleavage of adjacent hydroxyl groups of hydrocarbons occurs, accompanied by

[0045] In this embodiment, coupling to the carrier protein involves the lysyl transfer of the protein. For example, conjugation can be achieved by reductive amination via direct amination of any group. of sodium cyanoborohydride, optionally in the presence of nickel for aqueous conjugation. This is done by reacting a mixture of activated polysaccharides and carrier proteins with such a reducing agent. The conjugation reaction was carried out in aqueous solution or in the presence of dimethyl sulfoxide (DMSO). For example, U.S. Patent Application Publication US2015 / 0231270 and U S2011 / 0195086 and European Patent EP0471177B1. Addition of a reducing agent, such as sodium borohydride, caps the unreacted aldehyde. Conduct the training.

[0046] Reductive amination involves two steps: (1) oxidation of polysaccharides to form reactive aldehydes; (2) Reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein Prior to oxidation, the polysaccharide is cleaved into small sized fragments. Mechanical methods (e.g., homogenization) or chemical hydrolysis may be used. Chemical hydrolysis can be carried out with acetic acid. The oxidation step involves the reaction with periodate. In the context of the present invention, the term "periodate compound" refers to a compound containing periodate. The term includes both periodates and periodates, and the term also includes metaperiodate (IO4- ) and Orthoperiodate (IO6 5- ), and various salts of periodate compounds (e.g. In one embodiment, the capsular polysaccharide is In the presence of metaperiodate compounds, preferably in the presence of sodium periodate (NaIO4), In another embodiment, the capsular polysaccharide is oxidized in the presence of an orthoperiodate compound. The oxidation is preferably carried out in the presence of periodic acid.

[0047] In one embodiment, the oxidizing agent is an oxidizing agent for selectively oxidizing primary hydroxyls. In the presence of a stable nitroxyl or nitroxide radical compound, such as piperidine- N-oxy or pyrrolidine-N-oxy compounds (see, for example, International Patent Application Publication No. In the reaction, the actual oxidizing agent is a catalyst. In one embodiment, the stable nitroxy group is an N-oxoammonium salt in the nitroxy group. The nitroxide or nitroxide radical compounds are piperidine-N-oxy or pyrrolidine-N-oxy. In one embodiment, the stable nitroxyl or nitroxyl compound is an -N-oxy compound. The hydroxyl radical compound is TEMPO (2,2,6,6-tetramethyl-1-piperidinyl oxy) moiety or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) In one embodiment, the stable nitroxyl radical compound has a TEMP moiety. O or a derivative thereof. In one embodiment, the oxidizing agent is a molecule having an N-halo moiety. In one embodiment, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide Acid imide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloroisocyanuric acid 1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1, 3,5-Tribromo-1,3,5-triazinane-2,4,6-trione, diiodoiso Cyanuric acid and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-tri Preferably, the oxidizing agent is N-chlorosuccinimide. be.

[0048] In certain embodiments, the oxidizing agent is 2,2,6,6-tetramethyl-1-piperidinyl Oxy(TEMPO) free radical and N-chlorosuccinimide ( NCS) (described in International Patent Application Publication No. WO2014 / 097099). In one embodiment, the glycoconjugates from S. pneumoniae are prepared by: a) water The saccharide was converted to 2,2,6,6-tetramethyl-1-piperidinyloxy (TE MPO) and N-chlorosuccinimide (NCS) to produce activated saccharides and b) coupling the activated saccharide to a carrier protein having one or more amine groups. (This process is hereinafter referred to as "TEMP This is called "O / NCS-reductive amination."

[0049] Optionally, the oxidation reaction is quenched by adding a quenching agent. The agents include vicinal diols, 1,2-amino alcohols, amino acids, glutathione, and sulfur dioxide. Acid salts, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphorous salts acid salts or phosphoric acids (e.g., glycerin, ethylene glycol, propane-1,2-diol butane-1,2-diol or butane-2,3-diol, ascorbic acid) You can choose.

[0050] The second step of the conjugation process for reductive amination involves the use of a reducing agent: Stabilization by reduction of the imine (Schiff base) bond between activated polysaccharide and carrier protein The formation of a conjugate bond (so-called reductive amination) is a suitable reducing agent. Cyanoborohydrides (e.g., sodium cyanoborohydride) or sodium borohydride In one embodiment, the reducing agent is sodium cyanoborohydride.

[0051] In certain embodiments of the methods of the present invention, the reductive amination reaction is carried out in an aprotic solvent. (or a mixture of aprotic solvents). In one embodiment, the reduction reaction is , performed in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent If freeze-dried, the activated polysaccharide and capping agent are dissolved in DMSO or DMF solvent. In one embodiment, the aprotic solvent is DMSO. It is SO.

[0052] in DMSO after lyophilization using up to 5% sucrose at a 25x sucrose:Ps mass ratio. Optimal dissolution of the compound has been achieved. For example, International Patent Application Publication No. WO2017 / 0135 See 48. Streptococcus pneumoniae (S. pneumococcal) strains derived from serotypes 3, 8, and 24F In the case of polysaccharides, the polysaccharide is pre-coated in an aprotic solvent prior to protein conjugation. It was observed that higher levels of sucrose were required for sufficient dissolution. In embodiments, for these serotypes, sucrose concentrations greater than 5% in aqueous solution are used. In some embodiments, for these serotypes, 25 times more sucrose: Ps mass ratio, for example, at least 30 times, at least 35 times, or at least 40 times In some embodiments, the pre-lyophilized mass ratio of sucrose:polysaccharide is 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, 5 , 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 or more. Other sugars such as trehalose or mannitol can be used.

[0053] Protein conjugation, whether in aqueous or aprotic solvents With the presence of sodium chloride (NaCl) in the process, serotypes 15A, 16F, Streptococcus pneumoniae (S. pneumoniae) purified from 17F, 20, 23A, 24F, and 35B moniae) polysaccharides, reducing free protein levels and increasing the conjugate molecular weight and / or increase lysine consumption. Accordingly, the present invention provides a method for producing a polysaccharide-protein conjugate, comprising the steps of: The S. pneumoniae polysaccharide in the first solution is mixed with the protein in the second solution. and reacting the polysaccharide-protein conjugate with the protein to form a third solution, and causing a catalysis reaction to produce said polysaccharide-protein conjugate, wherein the third solution contains at least 1 mM salt.

[0054] Sodium chloride is used to freeze-dry polysaccharides and proteins prior to the conjugation reaction. Any point in the conjugation process from preparation to the conjugation reaction itself It can also be added at any point, for example, during the Schiff base reaction or sodium cyanoborohydride. In some embodiments, the compound may be added during reductive amination in the presence of ammonium. The sugars and proteins are freeze-dried separately and then the polysaccharide solution (first solution) or the protein solution (second solution) is added. In some embodiments, salt can be added to the protein solution (second solution) or both. The polysaccharide and protein are lyophilized from the same solution with added salt (i.e., first and second In some embodiments, the salt is added to the polysaccharide protein conjugate. Add to the solution in which the reaction is carried out (i.e., the third solution).

[0055] Other sodium salts, potassium salts, such as potassium chloride, lithium salts, magnesium salts, and Other salts such as calcium salts may be used. In the case of the Schiff base reaction, or in the reductive acyl group in the presence of sodium cyanoborohydride, During the cleavage reaction, 1 mM to 100 mM sodium chloride is added to the lysis solution. In embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM sodium chloride In some aspects of these embodiments, 100, 75, or 50 mM or less of Sodium chloride is used.

[0056] Once the reduction reaction is complete, there may be unreacted aldehyde groups remaining in the conjugate, which This is capped or quenched with a suitable capping or quenching agent. In one embodiment, the capping or quenching agent is , sodium borohydride (NaBH4). Suitable alternatives include Brönsted or or sodium triacetoxyborohydride or sodium borohydride in the presence of a Lewis acid boranes, such as pyridine borane, 2-picolinyl borane, Dimethylamine-borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe i P rN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine borane (P EMB) or borohydride exchange resin.

[0057] Glycoconjugates prepared using reductive amination in aprotic solvents are typically used in polyvalent pneumonia. It is used in coccal protein conjugate vaccines. In certain embodiments of a multivalent composition in which not all serotypes are produced, The reduction reaction of the remaining serotypes is carried out in aqueous solvents (e.g., PBS (phosphate buffered saline), MES (2-(N-morpholino)ethanesulfonic acid), HEPES, (4-(2-hydroxyethyl) (N-(2-acetyl)-1-piperazineethanesulfonic acid), Bis-Tris, ADA (N-(2-acetate) Amido)iminodiacetic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonyl) 3-Morpholino-2-hydroxypropanesulfonic 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-hydroxy (ethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-molybdenum-2-yl)- N-(2-hydroxyethyl)piperazinediaminetetraacetic acid), HEPPSO (N-(2-hydroxyethyl)piperazinediaminetetraacetic acid), N-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4- Bis(2-hydroxy-3-propanesulfonic acid), TEA (triethanolamine) , EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), Selected from Shin or HEPB, 6.0 to 8.5, 7.0 to 8.0, or 7.0 to 7.5 The reaction is carried out in a pH

[0058] In some embodiments, the glycoconjugates of the present invention have a molecular weight of 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, In other such embodiments, the polysaccharide has a molecular weight of 50 kDa. In other such embodiments, the polysaccharide has a molecular weight of 70 to 1,000 kDa. In other such embodiments, the polysaccharide has a molecular weight of 1 kDa to 900 kDa. In other such embodiments, the polysaccharide has a molecular weight of between 00 kDa and 800 kDa. In further such embodiments, , polysaccharides, 100kDa~1000kDa; 100kDa~900kDa; 100kDa ~800kDa;100kDa~700kDa;100kDa~600kDa;100k Da~500kDa;100kDa~400kDa;100kDa~300kDa;15 0kDa~1,000kDa;150kDa~900kDa;150kDa~800kDa a;150kDa~700kDa;150kDa~600kDa;150kDa~500 kDa;150kDa~400kDa;150kDa~300kDa;200kDa~1 ,000kDa;200kDa~900kDa;200kDa~800kDa;200k Da~700kDa;200kDa~600kDa;200kDa~500kDa;20 0kDa~400kDa;200kDa~300;250kDa~1,000kDa;2 50kDa~900kDa;250kDa~800kDa;250kDa~700kDa ;250kDa~600kDa;250kDa~500kDa;250kDa~400k Da;250kDa~350kDa;300kDa~1,000kDa;300kDa~ 900kDa;300kDa~800kDa;300kDa~700kDa;300kDa a~600kDa;300kDa~500kDa;300kDa~400kDa;400 kDa~1,000kDa;400kDa~900kDa;400kDa~800kDa ;400kDa~700kDa;400kDa~600kDa;or 500kDa~60 In certain embodiments using acid hydrolysis, the polysaccharide has a molecular weight of 10 kDa. 0kDa~200kDa, 25kDa~200kDa, 50kDa~200kDa, 10 kDa to 150kDa, 25kDa to 150kDa, or 50kDa to 150kDa It has a molecular weight.

[0059] Suitable alternative chemistries include the 1-cyano-4-dimethylaminopyridinium tetraphosphate esterification of saccharides. This involves activation with difluoroborate (CDAP) to form a cyanate ester. Thus, activated saccharides can be attached to carrier molecules either directly or via a spacer (linker) group. For example, the spacer can be: Cystamine or cysteamine can be used to obtain thiolated polysaccharides, which can be Maleimide-activated carrier proteins (e.g., with GMBS) or haloacetylated Carrier proteins (e.g., iodoacetamide [e.g., ethyl iodoacetamide] HCl] or N-succinimidyl bromoacetate or SIAB, or SIA, or SB Coupling to the carrier via a thioether linkage obtained after reaction with Preferably, a cyanate ester (optionally by CDAP chemistry) (produced) with hexanediamine or adipic acid dihydrazide (ADH) The amino-derivatized saccharide is then carboxylated via the carboxyl group on the protein carrier. Conjugation to carrier proteins using diimide (e.g., EDAC or EDC) chemistry Such conjugates are described in International Patent Application Publication No. WO93 / 15760 , WO95 / 08348 and WO96 / 29094; and Chu et al, 198 3, Infect. Immunity 40:245-256.

[0060] Other suitable techniques include carbodiimides, hydrazides, active esters, norborane, p-Nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU Many of these are described in International Patent Application Publication No. WO98 / 42721. Conjugation involves the reaction of the free hydroxyl groups of the saccharide with CDI. Response (Bethell et al, 1979, J. Biol. Chem. 25 4:2572-4;Hearn et al, 1981, J. Chromatog r. 218:509-18) followed by reaction with proteins to form carbamates A carbonyl linker can be used that can be formed by the formation of a bond. In this case, reduction of the anomeric terminus to a primary hydroxyl group, optionally a primary hydroxyl group Protection / deprotection of the primary hydroxyl group of the ... and coupling of the CDI carbamate intermediate with an amino group on the protein. .

[0061] After conjugation (reduction reaction and optional capping or quenching reaction) The glycoconjugates can then be purified (polysaccharide-protein complexes) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / dialysis, filtration, tangential flow filtration, ultrafiltration, precipitation / elution, column chromatography ( Ion exchange chromatography, multimodal ion exchange chromatography, DEAE, or Hydrophobic interaction chromatography (HCI) and depth filtration are included. See, for example, U.S. Pat. No. 6, In certain embodiments, the glycoconjugates are diafiltrated. Purification by ion exchange chromatography or size exclusion chromatography will be done.

[0062] One method for characterizing the glycoconjugates of the present invention is to characterize the glycoconjugates as they are conjugated to saccharides. This is due to the number of lysine residues in the carrier protein (e.g., CRM197). , which can be characterized as the extent of conjugated lysine (conjugated Evidence for lysine modification of carrier proteins is due to covalent linkage to polysaccharides. This can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation is the process of converting the carrier protein material used to make the conjugate. In a preferred embodiment, the number of recovered lysine residues is reduced compared to that of the complex sugar of the present invention. The conjugation degrees of quality are 2-15, 2-13, 2-10, 2-8, 2-6, and 2-5. , 2~4, 3~15, 3~13, 3~10, 3~8, 3~6, ​​3~5, 3~4, 5~15 , 5 to 10, 8 to 15, 8 to 12, 10 to 15, or 10 to 12. In the present invention, the degree of conjugation of the glycoconjugates 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 an embodiment, the degree of conjugation of the glycoconjugates of the present invention is 7 to 12. In one such embodiment, the carrier protein is CRM197.

[0063] The glycoconjugates of the present invention are characterized by the saccharide:carrier protein ratio (by weight). In some embodiments, the polysaccharide:carrier protein in the glycoconjugate may be The ratio (weight ratio) of the substances is 0.5 to 3.0 (for example, about 0.5, about 0.6, about 0.7, about 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 0.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In this embodiment, the saccharide:carrier protein ratio (weight ratio) is 0.5 to 2.0 , 0.5~1.5, 0.8~1.2, 0.5~1.0, 1.0~1.5 or 1.0~2. In yet another embodiment, the saccharide:carrier protein ratio (by weight) is 0. is 0.8 to 1.2. In a preferred embodiment, the capsular polysaccharide in the conjugate In some such embodiments, the ratio of carrier protein to carrier protein is 1 to 2. The carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the present invention Although not covalently conjugated to a protein, they are still glycoconjugates. The free saccharides may include the free saccharides present in the composition. Free saccharides may be either complex carbohydrates or non-complex carbohydrates. can be covalently associated with (i.e., non-covalently bound to or adsorbed or trapped on or by it).

[0064] In a preferred embodiment, the complex carbohydrates comprise about 50%, 45%, or Contains less than 40%, 35%, 30%, 25%, 20% or 15% free polysaccharides. In embodiments, the glycoconjugates contain less than about 25% free polysaccharides compared to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugates contain less than about 20% free polysaccharides compared to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugates contain less than about 15% free sugars compared to the total amount of polysaccharides. Contains isopolysaccharides.

[0065] Multivalent polysaccharide-protein conjugate vaccines The polysaccharide-protein conjugates obtained using the methods of the present invention are multivalent polysaccharide-protein conjugates. In certain embodiments, the antibody can be used in a protein conjugate vaccine. Multivalent polysaccharide-protein conjugate vaccines are a combination of free polysaccharides, polysaccharide-protein conjugates, and Streptococcus pneumoniae (S. pneumoniae) as either a component of the gate or a combination thereof oniae) serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 1 7F, 18B, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B , 23F, 24B, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35 F, and 38 containing S. pneumoniae capsular polysaccharides from one or more of In certain embodiments, the immunogenic composition comprises a multivalent pneumococcal vaccine. 2, 3, 4, 5, 6, 7, 8, 9 individually conjugated to the above carrier proteins , 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, 3 6, 37, 38, 39, 40, 41, 42, 43, or 44 Streptococcus pneumoniae (S. pneu Contains Streptococcus pneumoniae capsular polysaccharides from the serovar moniae Preferably, the composition is a mixture of certain blood Saccharides from the pure form are not conjugated to multiple carrier proteins.

[0066] After the individual glycoconjugates are purified, they are combined to formulate the immunogenic compositions of the present invention. These pneumococcal conjugates are manufactured by separate processes and It is bulk formulated into a dosage form.

[0067] Pharmaceutical / vaccine compositions The present invention further provides a method for treating a pulmonary arthritis with a steroid drug, comprising administering to a subject a pulmonary arthritis patient a pulmonary arthritis, or a pulmonary arthritis patient a pulmonary arthritis ... or a pulmonary arthritis patient a pulmonary arthritis, or a pulmonary arthritis patient a pulmonary arthritis, or a pulmonary arthr This product contains any combination of Streptococcus pneumoniae serotypes. Compositions, including pharmaceutical, immunogenic and vaccine compositions, consisting essentially of or consisting of The product is provided.

[0068] The preparation of polysaccharide-protein conjugates can be carried out using art-recognized methods. For example, the individual pneumococcal conjugates can be prepared in a physiologically acceptable form. The composition can be prepared by formulating it in a vehicle. Examples of such vehicles include water, buffered saline, and the like. Salt water, polyhydric alcohols (e.g., glycerin, propylene glycol, liquid polyethylene These include, but are not limited to, ethanol) and glucose solutions.

[0069] In a preferred formulation, the vaccine composition is prepared in an L-histidine buffer containing sodium chloride. It is formulated as a liquid.

[0070] As defined herein, an "adjuvant" is an agent that aids in the immunization of the immunogenic composition of the invention. Immunoadjuvants are substances that enhance immunogenicity when administered alone. Weak (e.g., no or weak antibody titers or cell-mediated immune responses elicited) enhance the immune response to an antigen, increase antibody titers to the antigen, and / or This may reduce the dose of antigen that is effective in generating an immune response. Bants are often given to boost immune responses and are well known to those skilled in the art. Suitable adjuvants for enhancing the effectiveness of the composition include, but are not limited to: However, it includes:

[0071] (1) Aluminum salts (alum), such as aluminum hydroxide and aluminum phosphate um, aluminum sulfate, etc.;

[0072] (2) Oil-in-water emulsion formulations (such as muramyl peptides (defined below) or bacterial cell wall components) with or without other specific immunostimulants, e.g., (a) 5% squalene, 0.5% Tw containing various amounts of MTP-PE. Microfluidizer (e.g., Model 110Y Microfluidizer) using a microscope (Microfluidics, Newton, MA). MF59 (WO 90 / 14837) incorporated into particles, (b) 10% squalene , 0.4% Tween 80, 5% Pluronic block polymer L121, and thr -Containing MDP and microfluidized into a submicron emulsion or vortexed (c) 2% squaraine. 0.2% Tween 80, and 3-O-deacetylated monophosphoryl lipid (d eaylated monophosphorylipid)A(MPL(trade name))( No. 4,912,094), trehalose dimycolate (TDM) and Cell wall skeleton (CWS), preferably MPL+CWS (Detox®) Ribi™ Adjuvant System (RAS) (Co) contains one or more bacterial cell wall components. rixa, Hamilton, MT); and (d) Montanide ISA;

[0073] (3) Saponin adjuvants, such as Quil A or STIMULON (trademark) QS-21 (Antigenics, Framingham, MA) (e.g., US See U.S. Patent No. 5,057,540) may be used or produced from such adjuvants. particles, e.g., ISCOMs (cholesterol, saponin, phospholipids, and amphiphilic proteins) Immunostimulating complexes formed by the combination of proteins and Iscomatrix (registered trademark) (having essentially the same structure as ISCOMs but containing no protein);

[0074] (4) Bacterial lipopolysaccharides, synthetic lipid A analogues, e.g., aminoalkylglucosamine phosphates Antiglucan phosphate (AGP), or its derivatives or analogues, which are available from Corixa One such AGP is available from U.S. Pat. No. 6,113,918. An example is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl 2- Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl] tetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino}-b -D-glucopyranoside, also known as 529 (formerly known as RC529) was known), formulated in aqueous form or as a stable emulsion;

[0075] (5) Synthetic polynucleotides, such as oligonucleotides containing one or more CpG motifs. Chid (U.S. Patent No. 6,207,646);

[0076] (6) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL -4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gamma interferon), granulocyte-macrophage colony-stimulating factor Macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor ( TNF), costimulatory molecules such as B7-1 and B7-2; and

[0077] (7) Complement, e.g., the trimer of complement component C3d.

[0078] In another embodiment, the adjuvant is a combination of two, three or more of the above adjuvants. The above mixture, e.g., SBAS2 (3-deacetylated monophosphoryl lipid A and QS2 1) is an oil-in-water emulsion that also contains 1.

[0079] Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglucose N-acetyl-normuramyl-L-alanine-2-(1′- 2'-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine Examples of such a device include, but are not limited to, MTP-PE.

[0080] In certain embodiments, the adjuvant is an aluminum salt. The adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine. Sodium salt adjuvants are well known in the art and are described, for example, in Harlow, E. nd D. Lane(1988;Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts.Resear (Ch in Immunology 143:489-493). The aluminum salts include, but are not limited to, hydrated alumina, alumina hydrate, alumina trihydrate, etc. ATH, aluminum hydrate, aluminum trihydrate, Alhydrogel (registered trademark) (Registered Trademark), Superfos, Amphogel®, Aluminum Hydroxide (I II), aluminum hydroxyphosphate sulfate (aluminum phosphate adjuvant (APA) )), amorphous alumina, alumina trihydrate or aluminum trihydroxy.

[0081] 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 using a thermocouple to achieve a monodisperse particle size distribution. Diafilter against water and steam sterilize.

[0082] In certain embodiments, commercially available Al(OH)3 (e.g., Denmark / Acc urate Chemical and Scientific Co., Westb Proteins were extracted using Alhydrogel or Superfos (Ury, NY). In another embodiment, the adsorption of the protein is performed by adjusting the pI (isoelectric pH) of the protein. ) and the pH of the medium. Proteins with low pI have higher affinity than proteins with high pI. Aluminum salts are also strongly adsorbed to positively charged aluminum ions. Builds a reservoir of Ag that is slowly released over time, preventing nonspecific activation of macrophages and complement activation and / or stimulate innate immune mechanisms (possibly due to stimulation of uric acid) For example, Lambrecht et al., 2009, See Curr Opin Immunol 21:23.

[0083] Monovalent bulk aqueous conjugates are typically mixed together and diluted. The batch is then sterile filtered. Aluminum phosphate adjuvant is added aseptically to determine the serotype. All Streptococcus pneumoniae (S. pneumococcales) except for 6B (diluted to the desired 8 μg / mL) niae) serotype, the intended final concentration was 4 μg / mL, and the final aluminum concentration was 2 The adjuvanted formulation batch is placed in a vial or syringe. can be.

[0084] In certain embodiments, the adjuvant comprises a CpG-containing nucleotide sequence, e.g., Cp G-containing oligonucleotides, particularly CpG-containing oligodeoxynucleotides (CpG O In another embodiment, the adjuvant is ODN 1826, which is Available from oley pharmaceutical group.

[0085] "CpG-containing nucleotides," "CpG-containing oligonucleotides," "CpG oligonucleotides" "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, Vacci ne 21:4297. In another embodiment, any other art-related term A CpG-containing oligonucleotide is any synthetic nucleotide. These include modified oligonucleotides with intercidal linkages, modified bases and / or modified sugar chains.

[0086] The use of CpG oligonucleotides is well known in the art, e.g., Sur 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 Carrier Syst. 23:89-110.

[0087] Administration / Dosage The compositions and formulations described herein allow the vaccine to be administered by systemic or mucosal routes. and for use in protecting or treating humans susceptible to infection, e.g., pneumococcal infection, by For example, the compositions and formulations described herein can be administered to humans in an immunologically effective amount. Streptococcus pneumoniae ( In a method for eliciting an immune response to S. pneumoniae capsular polysaccharide conjugates, In another example, the compositions and formulations described herein can be administered to humans immunologically. an immunologically effective amount of an immunogenic composition or formulation described herein for use in a method of vaccinating a human against pneumococcal infection, comprising administering can.

[0088] The optimal amount of components for a particular vaccine involves observing an appropriate immune response in the subject. For example, in another embodiment, the present invention can be confirmed by standard tests for the detection of HIV-1. Dosages for vaccination are determined by extrapolation from animal studies to human data. In embodiments, dosages are empirically determined.

[0089] An "effective amount" of a composition of the present invention is an amount that, upon subsequent challenge, inhibits the growth of a microorganism, such as Streptococcus pneumoniae ( The likelihood or severity of infection with S. pneumoniae It refers to the dose required to induce antibodies that intentionally reduce the level of vasopressin.

[0090] Methods of using the compositions and formulations described herein include the treatment of microorganisms, such as Streptococcus pneumoniae (S. The major clinical syndrome caused by P. pneumoniae is invasive infection (meningitis) , pneumonia, and bacteremia) and non-invasive infections (acute otitis media and sinusitis) It can be used for prevention and / or reduction.

[0091] Administration of the compositions and formulations described herein can be by intramuscular, intraperitoneal, intradermal, or subcutaneous routes. or mucosal administration to the oral / alimentary, respiratory or genitourinary tract. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (e.g., pneumonia). The nasopharyngeal passage of cocci can be more effectively inhibited, thereby attenuating infection at an early stage. eye).

[0092] The amount of conjugate in each vaccine dose was determined to be sufficient to induce an immunoprotective response without significant adverse effects. Such an amount may vary depending on the serotype of pneumococcus. For polysaccharide conjugates, each dose is 0.1-100 μg, especially 0.1-10 μg. g, more particularly 1-5 μg of each polysaccharide. For example, each dose may contain 100, 150, or 200 μg of each polysaccharide. , 200, 250, 300, 400, 500 or 750 ng or 1, 1.5, 2, 3 , 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18 , 20, 22, 25, 30, 40, 50, 60, 70, 80, 90 or 100 μg It may include.

[0093] The optimal amount of components for a particular vaccine involves observing an appropriate immune response in the subject. For example, in another embodiment, the present invention can be confirmed by standard tests for the detection of HIV-1. Dosages for vaccination are determined by extrapolation from animal studies to human data. In another embodiment, dosages are empirically determined.

[0094] In one embodiment, the dose of aluminum salt is 10, 15, 20, 25, 30, 50 , 70, 100, 125, 150, 200, 300, 500 or 700 μg, or 1 , 1.2, 1.5, 2, 3, 5 mg or more. The doses of aluminum salts given are per μg of recombinant protein.

[0095] Generally, each 0.5 mL dose contains 2 μg of pneumoniae strains but 4 μg of serotype 6B polysaccharide. S. pneumoniae polysaccharide; approximately 32 μg of CRM197 carrier protein (e.g., 32 μg ± 5 μg, ± 3 μg, ± 2 μg, or ± 1 μg); 0.125 mg Elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride The sodium chloride concentration is approximately 100mg / mL. 150 mM (e.g., 150 mM ± 25 mM, ± 20 mM, ± 15 mM, ± 10 mM, or ±5 mM), and the L-histidine buffer is about 20 mM (e.g., 20 mM ±5 mM). M, ±2.5 mM, ±2 mM, ±1 mM, or ±0.5 mM).

[0096] According to the methods of using the compositions or formulations described herein, and in one embodiment In certain embodiments, the human patient is an infant (under 1 year old), a toddler, or a young child. The children are toddlers (approximately 12-24 months) or toddlers (approximately 2-5 years). In some embodiments, the human patient is an elderly patient (>65 years old). The compositions of the present invention may also be used in older children, It is also suitable for use in adolescents and adults (e.g., aged 18-45 or 18-65).

[0097] In one embodiment of the method of using the compositions or formulations described herein, the composition or formulation The formulation is administered as a single inoculation. In another embodiment, the composition or formulation is administered as a single inoculation. The composition may be administered once, three or four times or more, with sufficient time between doses. or formulations at 1, 2, 3, 4, 5, or 6 month intervals, or any combination thereof. The immunization schedule may be similar to that designed for pneumococcal vaccines. For example, the infection caused by Streptococcus pneumoniae (S. pneumoniae) The routine schedule for infants and toddlers for common invasive diseases is: 2, 4, 6 and 12 to 15 months of age. The product is administered in a series of four doses at ages 2, 4, 6 and 12-15 months.

[0098] The compositions described herein also comprise a Streptococcus pneumoniae (S. pneumoniae) derived It may also contain one or more proteins. moniae proteins are described in WO 02 / 083855 and WO 02 / 083856. Including those identified in No. 02 / 053761.

[0099] formulation The compositions described herein are administered to a subject by one or more methods known to those of skill in the art, for example, parenterally, It can be administered transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, or intraperitoneally. It can be formulated according to the need.

[0100] In one embodiment, the compositions described herein are administered by epidermal, intramuscular, or intravenous injection in a liquid formulation. It is administered by intravenous, intraarterial, subcutaneous, or respiratory intramucosal injection. This includes liquids.

[0101] The compositions may be formulated as single-dose vials, multi-dose vials, or pre-filled syringes. It is possible.

[0102] In another embodiment, the composition is administered orally and is therefore in a form suitable for oral administration, i.e. They are formulated as solid or liquid preparations. Solid oral formulations include tablets, capsules, and These include pills, granules, pellets, etc. Liquid oral preparations include solutions, suspensions, dispersions, emulsions, etc. Includes fluids, oils, etc.

[0103] Pharmaceutically acceptable carriers for liquid formulations include aqueous or non-aqueous solutions, suspensions, emulsions, and the like. Examples of the non-aqueous solvent include propylene glycol and polyethylene glycol. and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcohol, and the like. Oil-based / aqueous 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 , sunflower oil, fish liver oil, other fish oils, or lipids derived from milk or eggs.

[0104] Pharmaceutical compositions can be isotonic, hypotonic, or hypertonic. In this case, it is preferred that the pharmaceutical composition for infusion or injection is essentially isotonic when administered. Therefore, during storage, the pharmaceutical composition may preferably be isotonic or hypertonic. If the drug composition is hypertonic during storage, it should be diluted to an isotonic solution before administration. can be done.

[0105] The isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a sugar. Examples of ionic isotonic agents include NaCl, CaCl, KCl, and Mg Examples of non-ionic isotonic agents include, but are not limited to, Cl2. Contains sucrose, trehalose, mannitol, sorbitol and glycerol, It is not limited to:

[0106] Preferably, the at least one pharmaceutically acceptable additive is a buffering agent. For some purposes, for example, when the pharmaceutical composition is for infusion or injection, the composition may contain a softening agent. It is often desirable to include a buffer, which can provide a solution in the range of 4-10, e.g. It has the ability to buffer a pH of 5 to 9, for example 6 to 8.

[0107] Buffers include, for example, TRIS, acetic acid, glutamine, lactic acid, maleic acid, tartaric acid, and phosphate. , citric acid, carbonic acid, glycinic acid, histidine, glycine, succinic acid and triethanolamine The buffer may be selected from the group consisting of:

[0108] Furthermore, buffering agents may be used, for example, to provide a non-steroidal anti-inflammatory agent, especially when the pharmaceutical formulation is for parenteral use. The buffer may be selected from USP compatible buffers for oral use. For example, the buffer may be Basic acids (acetic acid, benzoic acid, gluconic acid, glyceric acid and lactic acid); dibasic acids (aconitic acid Acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid and tartaric acid ), polybasic acids (citric acid and phosphoric acid); and bases (ammonia, diethanolamine, glycine, triethanolamine and TRIS).

[0109] Parenteral vehicles (for subcutaneous, intravenous, arterial, or intramuscular injection) include sodium chloride solution, phosphate Gel dextrose, dextrose and sodium chloride, lactated Ringer's solution and volatile oil Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., phosphate Examples include surfactants and other pharmaceutically acceptable carriers. These are sterile liquids (such as water and oils) with or without the addition of adjuvants. Generally, water, saline, aqueous dextrose and related sugar solutions, glycols (propylene glycol) glycol or polyethylene glycol), polysorbate-80 (PS-80), Polysorbate-20 (PS-20) and Poloxamer 188 (P188) are preferred. A liquid carrier (especially for injectable solutions). Examples of oils are those of animal, vegetable or synthetic origin. , e.g., peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other fish oils, or It is a lipid derived from milk or eggs.

[0110] The formulation may also include a surfactant. Preferred surfactants include polyoxyethylene Styrene sorbitan ester surfactants (commonly referred to as Tween), especially PS -20 and PS-80; ethylene oxide (EO), propylene oxide (PO) and / or or copolymers of butylene oxide (BO) (sold under the trade name DOWFAX®); For example, linear EO / PO block copolymers; octoxynol (which contains repeating ethoxy groups); The number of (oxy-l,2-ethanediyl) groups can be varied, and octoxynol- 9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) (Octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); phenol ethoxylates, such as the Tergitol (trade name) NP series; Polyoxyethylene fats derived from ethyl, cetyl, stearyl and oleyl alcohols Aliphatic ethers (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly S PAN), such as sorbitan trioleate (Span 85) and sorbitan trioleate (Span 85). This emulsion contains, but is not limited to, lauric acid ... A particularly preferred surfactant is PS-20 or PS-80.

[0111] Mixtures of surfactants (e.g., PS-80 / Span 85 mixtures) can also be used. Also, polyoxyethylene sorbitan ester (polyoxyethylene sorbitan monooleate (PS-80)) and octoxynol (t-octylphenoxypolyethoxy Another useful combination is ethanol (Triton X-100). Laureth 9 + Polyoxyethylene Sorbitan Ester and / or Octoxynol Includes.

[0112] The preferred amount of surfactant is: polyoxyethylene sorbitan ester (e.g., PS-80) ) is 0.01 to 1% by weight / volume, in particular about 0.1% by weight / volume; octyl- or nonylphenoxyethanol hydroxypolyoxyethanol (e.g., Triton X-100, or Triton X-100) Other cleaning agents) are 0.001 to 0.1 wt / vol %, especially 0.005 to 0.02 wt / vol Amount / volume %: Polyoxyethylene ether (such as Laureth 9) 0.1 to 20 weight / volume %, preferably 0.1 to 10% by weight / volume, in particular 0.1 to 1% by weight / volume or about 0.5% by weight / volume Amount / volume %.

[0113] In certain embodiments, the composition contains 250 μg / mL of APA (aluminum phosphate). L-histidine (20 mM), saline at pH 5.8, together with ammonium adjuvant (150 mM) and 0.2% weight / volume PS-20. In formulations that suppress aggregation during simulated manufacturing and transportation using primary packaging, PS-20 or PS-8 0 may be present and may range from 0.005 to 0.1% weight / volume. , up to 44 S. pneumoniae polysaccharide serotypes / L-histidine combining a mixture of ethanol, sodium chloride, and PS-20; and then subjecting the mixture to an antibacterial It consists of combining APA and sodium chloride with or without preservatives. do.

[0114] When surfactant selection needs to be optimized for different drug products and drug substances Multivalent vaccines containing 15 or more S. pneumoniae polysaccharide serotypes In the case of cutin, PS-20 and P188 are preferred. The choice of chemistry used can also affect the stability of the formulation. In particular, as exemplified below, or DMSO solvent and combined pneumococcal polysaccharide-protein in a multivalent composition. The stability of the polymer conjugates varies greatly depending on the specific surfactant system used in the formulation. Show the difference.

[0115] For the formulations described herein, poloxamers typically range from 1,100 Da to 17,400 Da. a, 7,500 Da to 15,000 Da or 7,500 Da to 10,000 Da The poloxamer is selected from poloxamer 188 and poloxamer 407. The final concentration of poloxamer in the formulation of the present invention is 0.001 to 5% by weight. Poloxamer-containing surfactant systems are also available. In addition, it must contain a polyhydric alcohol. The alcohol is propylene glycol and is used at a final concentration of 1-20% weight / volume. In certain embodiments, the polyhydric alcohol is polyethylene glycol 400. and is included at a final concentration of 1 to 20% weight / volume.

[0116] Suitable polyhydric alcohols for the formulation include polymeric polyhydric alcohols, particularly polyether diols. glycols, such as propylene glycol and polyethylene glycol, polyethylene glycol Propylene glycol monomethyl ethers, including but not limited to: Polyols are available in a range of monomer molecular weights from about 425 Da to about 2,700 Da. Ethylene glycol and polyethylene glycol monomethyl ether are from about 200 Da They are also available in molecular weight ranges of about 35,000 Da, e.g., PEG200, PEG3 00, PEG400, PEG1000, PEGMME550, PEGMME600, PE Including, but not limited to, GMME2000, PEGMME3350 and PEGMME4000 The preferred polyethylene glycol is polyethylene glycol 4 The final concentration of polyhydric alcohol in the formulation is 1-20% weight / volume or 6-20%. It can be in % weight / volume.

[0117] The formulation also includes a pH buffered saline solution. Buffering agents include, for example, Tris, acetate, glutathione, PEG, PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, glutamin, lactic acid, maleic acid, tartaric acid, phosphoric acid, citric acid, carbonic acid, glycinic acid, L-histamine Thiamine, glycine, succinic acid, HEPES (4-(2-hydroxyethyl)-1-piperazine diethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), M From ES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer The buffer may be selected from the group consisting of 4 to 10, 5.2 to 7.5, or 5.8 to 7 In certain embodiments, the buffering agent has the ability to buffer the solution to a pH in the range of 0.0. phosphate, succinate, L-histidine, MES, MOPS, HEPES, acetate or quercetin The buffering agent is selected from the group consisting of phosphates. The buffering agent may further be, for example, a phosphate salt, especially for pharmaceutical preparations. If the formulation is intended for parenteral use, it may be selected from USP compatible buffers for parenteral use. The concentration of the buffer solution ranges from 1 mM to 50 mM or from 5 mM to 50 mM. In one embodiment, the buffering agent is L-histidine at a final concentration of 5 mM to 50 mM, or In certain embodiments, L-histidine is present at a final concentration of 10 mM to 10 mM succinate. 20mM±2mM.

[0118] Although saline solutions (i.e., solutions containing NaCl) are preferred, other salts suitable for formulation include: , CaCl2, KCl, and MgCl2, and combinations thereof. Sucrose, trehalose, mannitol, sorbitol and glycerin ( Non-ionic isotonicity agents such as, but not limited to, benzodiazepines ... and benzoylcholinesterase inhibitors can be used in place of salts. Suitable salt ranges include 25 mM to 500 mM or 40 mM to 170 mM. In one embodiment, the saline solution is NaCl, It may be present at a concentration of 20 mM to 170 mM.

[0119] In a preferred embodiment, the formulation comprises an L-histidine buffer containing sodium chloride. nothing.

[0120] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent is delivered intravenously. It can be administered using injection, transdermal patch, liposomes, or other modes of administration. In embodiments, the polymeric material is used, for example, in a microsphere or implant. will be done.

[0121] The compositions described herein may comprise one or more Streptococcus pneumoniae (S. pneumoniae)-derived antibodies. Proteins of Streptococcus pneumoniae (S. pneumoniae) may also be included. Examples of proteins are described in International Patent Application Publication Nos. WO02 / 083855 and WO02 Including those confirmed in / 053761.

[0122] Analysis method Molecular weight and concentration of the conjugate using HPSEC / UV / MALS / RI assay analysis Inject the conjugate sample and perform high performance size exclusion chromatography (HPSEC) Detection is by a series of ultraviolet (UV), multi-angle light scattering (MALS) and refractive index. Calculate protein concentration from UV280 using the extinction coefficient (RI) detection. d is the change in refractive index of the solution due to a change in solute concentration reported in mL / g Using the n / dc coefficient, the RI signal (contributions from both proteins and polysaccharides) is used to calculate the multi- The average molecular weight of the sample is determined by the measured concentration and the total sugar concentration of the sample peak. Light scattering data was used to analyze the data using Astra software (Wyatt Technology Corporation, Santa Barbara, CA). For small molecules, there are many forms of molecular weight averages, such as number average molecular weight Mn, weight average molecular weight Mn, Average molecular weight Mw, and z-average molecular weight Mz (Molecules, 2015, 20: 10313-10341). Unless otherwise specified, as used throughout this specification, The term "molecular weight" refers to the weight average molecular weight.

[0123] Conjugation time as a measure of the number of covalent bonds between the polysaccharide and the carrier protein Measurement of lysine consumption in proteins. Waters AccQ-Tag amino acid analysis (AAA) was used to identify conjugates The degree of conjugation in the sample is measured. The sample is hydrolyzed using phase acid hydrolysis to separate the carrier protein into its component amino acids. The free amino acids are decomposed into 6-aminoquinolyl-N-hydroxysuccinimide. The resulting derivatized sample was then derivatized with benzoyl carbamate (AQC). Analyze using UPLC with UV detection on a C18 column. The lysine consumption during conjugation (i.e., Lysine loss (Lysine loss) is calculated by dividing the average measured amount of lysine in the conjugate by the amount of lysine in the starting protein. It is calculated by the difference between the predicted amount.

[0124] Free polysaccharide test First, the free protein and conjugate were dissolved in deoxycholate (DOC) and hydrochloric acid. The free polysaccharides in the conjugate sample (i.e., CRM197 and Next, the precipitate was removed by filtration, and the filtrate was analyzed by HPSE. The free polysaccharide concentration was analyzed by C / UV / MALS / RI. Calculate as a percentage of total polysaccharides as determined by UV / MALS / RI.

[0125] Free Protein Test Free polysaccharide in the conjugate sample, polysaccharide-CRM197 conjugate, and free CRM197 was analyzed by capillary electrokinetic chromatography in micellar electrokinetic chromatography (MEKC) mode. The samples were separated by electrophoresis in 25 mM borate, 100 mM SDS, pH 7.0. Mix with MEKC running buffer containing H9.3 and pre-conditioned The separation was performed on a treated fused silica capillary. The separation was monitored at 200 nm to determine the free CR CRM197 is quantified using a CRM197 standard curve. Free protein results are compared with HPS as a percentage of total protein content determined by EC / UV / MALS / RI procedure Report.

[0126] Although various embodiments of the present invention have been described above with reference to the accompanying description and drawings, it should be understood that It should be noted that the present invention is not limited to the embodiments described in detail, and that those skilled in the art will appreciate the following detailed description of the invention. Various modifications and variations are possible without departing from the scope or spirit of the present invention as defined in the claims. Corrections can be made.

[0127] The following examples illustrate but do not limit the invention. [Example]

[0128] Example 1: Production of S. pneumoniae capsular polysaccharide Methods for culturing pneumococci are known in the art. See, e.g., Chase, 1967, Me thods of Immunology and Immunochemistry 1:52. Methods for producing pneumococcal capsular polysaccharides are also known in the art. Reference is made to patent EP0497524B1. The process described below is also described in European patent EP0497524B1. All pneumoniae except as specifically modified, according to the method described in 7524B1. Applicable to cocci serotypes.

[0129] Isolates of pneumococcal serotypes 3, 8, and 12F were obtained from the University of Pennsylvania (Rob Dr. ert Austrian). Pneumococcus serotypes 15A, 16F, 23A, 24F, 3 The 5B isolate was obtained from the Merck Culture Collection. Isolates of serotypes 23B and 31 were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). The isolate of Streptococcus pneumoniae serotype 17F was obtained from the FDA Office of Biologics (J Pneumococcal serotype 20 isolates were obtained from ATCC. If necessary, based on the Quelling reaction using specific antisera, Subtypes can be differentiated. See, e.g., U.S. Patent No. 5,847,112. The resulting isolate was cultured in a medium containing hemin-free soy peptone, yeast extract, and glucose. Further cloning was performed by plating the cells sequentially in two stages on agar plates consisting of animal component-free medium containing Soy peptone, yeast extract, HEPES, sodium chloride, bicarbonate An animal component-free medium containing sodium, potassium phosphate, glucose, and glycerin was used. Clonal isolates for each serotype were further expanded in liquid culture containing pre-master isolates. We created a cell bank.

[0130] The production of each serotype of pneumococcal polysaccharide involves cell growth and batch production fermentation followed by chemical depletion. The study consisted of thawing cell bank vials from each serotype followed by downstream purification. soybean peptone or soybean peptone ultrafiltrate, yeast extract or yeast extract ultrafiltrate solution, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, and glucose Growth in shake flasks or culture bottles containing sterile, animal-component-free growth medium containing Cell growth cultures were grown in closed shake flasks or bottles under temperature and agitation control. Gas exchange was minimized during incubation. The culture densities measured by optical density at 600 nm were determined. Once this is achieved, a portion of the cell growth culture is added to soy peptone or soy peptone ultrafiltrate. , yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate and glutamic acid The culture was transferred to a production fermenter containing sterile, animal component-free growth medium containing the course. pH, pressure and agitation were controlled. No sparging was used and airflow overlay was also controlled. Ta.

[0131] Once the glucose is almost exhausted, the batch is quenched by adding phenol, a chemical quencher. Fermentation was terminated by adding pure phenol to a final concentration of 0.8-1.2%. The cells were inactivated to release the capsular polysaccharides from the cell wall. Primary inactivation was performed in a fermenter for a specified time. After the first inactivation, the batch is transferred to a separate vessel. where it is kept under controlled temperature and agitation for an additional specified time. This was achieved by microbial plating techniques or by measuring the phenol concentration and This was confirmed by one of the specified time validations. The inactivated broth was then purified.

[0132] Purification of Ps The purification of pneumococcal polysaccharides involves several centrifugation, depth filtration, and concentration / diafiltration steps. All procedures were performed at room temperature unless otherwise noted. Ta.

[0133] Inactivated broth from a fermenter culture of S. pneumoniae was ionic polymers (e.g., BPA-1000, Petrolite, Tretolite) " and "Spectrum 8160" and poly(ethyleneimine), "Millipor The cationic polymer aggregated the impurity protein with α-pDADMAC. After the flocculation step and maturation period, the flocculated solids were centrifuged and washed multiple times. The purified broth was concentrated to a concentration of 100 kDa to 500 kDa. Diafiltration was performed using a MWCO (molecular weight cut-off) filter. The filtration was performed using Tris, MgCl2 buffer and sodium phosphate buffer. Residual nucleic acids and proteins were removed by diafiltration. .

[0134] Denaturation of polysaccharides in sodium acetate and phenol, alcohol and / or isopropanol Further impurity removal was achieved by reprecipitation with sodium acetate during the phenol precipitation step. sodium phosphate / sodium phosphate saline buffer and phenol (liquefied phenol or solid The diafiltered retentate was then added to the diafiltered retentate. Coal fractionation was carried out in two stages. In the first stage, a low percentage of alcohol was added to the precipitate. This allowed the cell debris and other undesirable impurities to precipitate, while the crude polysaccharide remained in solution. The impurities were removed by a depth filtration step. Additional isopropanol or denatured alcohol was then added. The polysaccharide was recovered from the solution by adding ethanol to the batch. The precipitated polysaccharide pellet was collected by centrifugation. The mixture was collected by grinding, dried to a powder, and stored frozen at -70°C.

[0135] Example 2: General conjugation method Polysaccharide size reduction and oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water and filtered through a 0.45 micron filter. Unless otherwise specified, the polysaccharide was homogenized to reduce the polysaccharide molecular weight. Homogenization pressure and homogenizer The number of passes was controlled to serotype-specific settings (150–1000 bar; 4–7 passes). passing).

[0136] The reduced size polysaccharide was filtered through 0.2 microns, concentrated, and purified to 5 kDa or 10 kDa NM. Diafiltration was performed against distilled water using a WCO tangential flow ultrafiltration membrane.

[0137] The polysaccharide solution was then adjusted to 22°C and pH 5 with sodium acetate buffer to activate The reduction in polysaccharide size due to cleavage was minimized.

[0138] The purified polysaccharide was prepared for conjugation, i.e., using sodium metaperiodate oxidation. (Anderson et al., 1986, J. Immuno l. 137:1181-1186; and U.S. Patent Application Publication No. US201101950 (See 86). 100 mM sodium metaperiodate solution was added to 50 mM sodium acetate of the polysaccharide. The amount of sodium metaperiodate added was approximately 0.1 to 0.5 molar equivalents. Serotype-specific, ranging from 1 mole of sodium metaperiodate / polysaccharide repeating unit The desired level of polysaccharide activation was achieved (per mole of polysaccharide repeating unit). (moles of aldehyde per 1000 ml) The samples were incubated for the specified time in the dark. It was mixed throughout.

[0139] The resulting activated product was filtered using a 5 kDa or 10 kDa NMWCO tangential flow ultrafiltration membrane. Diafiltrate against 10 mM potassium phosphate, pH 6.4, and then against distilled water. All serum samples were diafiltered against water and then further diafiltered against water. Ultrafiltration of the mold was carried out at 2-8°C.

[0140] Conjugation Pseudomonas fluorescens (Pseudomonas fluorescens) was isolated according to a previously reported method (WO2012 / 173876A1). Purified CRM197 obtained by expression in C. fluorescens was analyzed using a 5 kDa a Dialysis of 2 mM phosphate pH 7.2 buffer using a NMWCO tangential flow ultrafiltration membrane. The solution was filtered through a 0.2 micron filter.

[0141] Activated polysaccharides were prepared by freezing at sucrose concentrations of 0.5–30% wt / vol and 1–6 mg Ps / mL. CRM197 was formulated for freeze-drying at 6 mg Pr / m2 at a sucrose concentration of 1% w / v. It was formulated for lyophilization at 1 L.

[0142] The formulated Ps and CRM197 solutions were lyophilized separately. The RM197 was separately redissolved in an equal volume of DMSO. The polysaccharides were mixed to achieve the desired polysaccharide concentration and polysaccharide:CRM197 mass ratio. The polysaccharide:CRM197 ratio in the resulting conjugate was controlled by cyanohydride. Sodium boronate (1 mole / 1 mole of polysaccharide repeat unit) was added and the conjugation was The reaction was allowed to proceed at 22°C for the indicated period.

[0143] Reduction with sodium borohydride After the conjugation reaction, sodium borohydride (2 moles / polysaccharide repeat unit) was added. The batch was stirred at about 4° C. with about 0.025% (wt / vol) polysorbate The solution was diluted with 150 mM sodium chloride in 10 mL of 100 mL of potassium phosphate buffer. In addition, the pH was neutralized.

[0144] Final Filtration and Product Storage The conjugate was then filtered using a 300 kDa NMWC membrane at approximately 4°C with 0.05% (weight dialyzed against 150 mM sodium chloride with (volume / volume) polysorbate 20 or Using a 30 kDa NMWC tangential flow ultrafiltration membrane, the filtration was carried out using 25 mM potassium phosphate pH 7. Diafilter against 150 mM sodium chloride containing or without sodium chloride, then concentrate and filtered at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane to a concentration of 0.015% ( (wt / vol) 10 mM histidine / 150 mM sodium chloride with polysorbate 20 The remaining batch was diafiltered against 10 mM HCl (pH 7.0). Stidin / 150 mM sodium chloride (pH 7.0) filtered and 0.2 micron filtered The final conjugate solution was aliquoted and frozen at ≦−60°C.

[0145] Example 3: Acid hydrolysis of polysaccharides from serotypes 12F, 23A, 24F and 31 Protein synthesis of pneumococcal polysaccharides by reductive amination in aprotic solvents such as DMSO. Conjugation to proteins has been previously reported. Typically, activated polysaccharides (Ps) and protein (Pr) were lyophilized and resuspended in DMSO, then mixed and diluted with water. Incubation with sodium cyanoborohydride and sodium borohydride Conjugation was performed after mechanical size reduction (e.g., homogenization) of the polysaccharide. Therefore, the Ps molecular weight is reduced by oxidation to obtain a constant Ps size for conjugation. For many pneumococcal serotypes, mechanical size reduction and oxidative Size, lysine consumption, free polysaccharides, and free proteins by Ps conjugation However, it is difficult to obtain a conjugate that satisfies the desired properties for some serotypes. Regarding this, even after optimizing the process parameters, the process It has been found that adjugate properties are difficult to achieve.

[0146] Ps size decrease Purified pneumococcal capsular polysaccharide powders from serotypes 23A, 24F, and 31 were dissolved in water. The experimental arms were treated mechanically by homogenization or chemically by acid hydrolysis to The homogenization pressure and the number of passes through the homogenizer were adjusted to reduce the molecular weight of the serotype Ps. The batch was heated to 90°C and controlled at a constant pressure (150-1000 bar; 4-7 passes). Bring to 92°C, add concentrated acetic acid to a final concentration of 200 mM, then incubate for up to 90 minutes. After the incubation period, the solution was added to concentrated potassium phosphate. The batch was prepared by adding pH 7 buffer to a final concentration of 400 mM and cooling to ≤22°C. The reduced size polysaccharide was filtered through 0.2 microns, concentrated, and purified to 5 kDa or 10 kDa. a Diafiltered against water using a NMWCO tangential flow ultrafiltration membrane.

[0147] The polysaccharide was conjugated according to the method described in Example 2.

[0148] The experimental conditions and results are summarized in Table 1.

[0149] Table 1. Streptococcus pneumoniae serotypes 23A, 24F, and 31 Summary of experimental arm on Ps size reduction by homogenization and acid hydrolysis [Table 1]

[0150] As can be seen in Table 1, the size reduction by acid hydrolysis is , high lysine loss (serotype 23A), low free Ps (serotypes 24F and 31), or low A means of achieving free Pr (serotypes 23A, 24F and 31) was provided. In the case of 24F, higher free protein levels in homogenizations were associated with aggregated forms. This may have contributed to the higher measured conjugate Mw levels. There is a possibility.

[0151] Without being bound by any particular theory, the data suggest that the The relatively low molecular weight (preferentially less than 150 KDa) of the oxidized polysaccharides conjugates This suggests that the effect of ATP on the ATP synthesis was beneficial (less free P or free Pr) in improving the ATP synthesis. Another size reduction process (e.g., by acid hydrolysis) can be used to reduce this preferred size. It is believed that it is possible to achieve similar conjugation effects by using polysaccharides in the same size range. can be obtained.

[0152] Effect of acid form on the acid hydrolysis of serotype 12F while maintaining a constant pH. To confirm whether the acid type affected the polysaccharide size, acid hydrolysis using hydrochloric acid was performed using acetic acid. Purified pneumococci from S. pneumoniae serotype 12F were compared. Capsular polysaccharide powder was dissolved in water and 0.45 μm filtered. The batch was adjusted to 2.5 g Ps / L. The diluted solution was divided into two arms. For both arms, they were first heated to 80°C. Acid hydrolysis was performed by adding acid to maintain a similar pH in both arms. To one arm, glacial acetic acid was added to a final concentration of 200 mM, pH 2.6. 1N hydrochloric acid was added to the arms to a final concentration of 2.5 mM, pH 2.7. The mixture was incubated for 155 minutes. After the incubation period, concentrated potassium phosphate solution was added. The arms were neutralized by adding H buffer to a final concentration of approximately 400 mM and cooling to 4°C. The results are shown in Table 2.

[0153] Table 2: Acid hydrolysis of S. pneumoniae 12F polysaccharide using hydrochloric acid Solution [Table 2]

[0154] Example 4: From S. pneumoniae serotypes 3, 8 and 24F Effect of sucrose:polysaccharide mass ratio on the dissolution of polysaccharides in DMSO Alternatives for preparing polysaccharides for protein conjugation in aprotic solvents Typically, the polysaccharide and protein solutions are freeze-dried. , sucrose concentration 5% weight / volume (50 mg sucrose / mL) with 6 mg Ps / mL aqueous solution Formulating activated polysaccharides (Ps) for lyophilization in DMSO allows for reconstitution and Lyophilizates suitable for conjugation were obtained. For serotypes 3, 8, and 24F , this formulation (6 mg Ps / mL, 50 mg sucrose / mL, sucrose:Ps mass ratio = 8.3 ) was found to produce a freeze-dried product that is insoluble in DMSO.

[0155] To optimize activated polysaccharide lyophilized formulations for dissolution in DMSO, The experiment was carried out. Activated polysaccharides were added to polypropylene containers at a wide range of polysaccharide concentrations (1 to 6 mg). The solutions were formulated at different concentrations (50-300 mg sucrose / mL) and sucrose concentrations (50-300 mg sucrose / mL). Freeze and dry to remove water, then add DMSO at ambient temperature with mixing to redissolve the polysaccharide. Whether or not the solution was dissolved satisfactorily was confirmed by visual observation.

[0156] The results for serotypes 3, 24F, and 8 are shown in Tables 3, 4, and 5, respectively.

[0157] Table 3. Serotype 3 lyophilized formulations and dissolution experiments [Table 3] TIFF0007815333000004.tif12170

[0158] Table 4. Serotype 24F lyophilized formulation and dissolution experiments [Table 4]

[0159] Table 5: Serotype 8 lyophilized formulations and dissolution experiments [Table 5]

[0160] For these serotypes, the range of polysaccharide concentrations examined (both lyophilized and dissolved) Complete dissolution was observed within the range of 0.1% to 1.0% of the polysaccharide concentration, but dissolution was dependent on both the polysaccharide and sucrose concentrations. Specifically, when the mass ratio of sucrose was less than 30 times that of polysaccharides, freeze-drying Subsequent dissolution in DMSO was not achieved. The most consistent dissolution results were observed for the sucrose mass This was observed when the ratio was at least 40 times that of the polysaccharide.

[0161] Several arms from the positive lysis conditions were successfully treated with CRM197 as described in Example 2. was conjugated to

[0162] Dissolution of Streptococcus pneumoniae (S. pneumoniae) polysaccharides from serotype 3 in DMSO Effect of sugar type and sugar:polysaccharide mass ratio on Effect of sugar type and sugar:polysaccharide mass ratio on the dissolution of lyophilized activated polysaccharide in DMSO An experiment was conducted to evaluate the effect of activated serotype 3 polysaccharide on the microbial activity of the bacteria. Either 0.5 or 6 mg Ps / mL, and a range of sugar concentrations (50 to 150 mg The sugars investigated were sucrose, trehalose, and mannitol. The solution was lyophilized to remove water, and then DMSO was added at ambient temperature with mixing to obtain a The sugar was redissolved. Whether it was dissolved well or not was confirmed by visual observation. The results are shown in Table 6. It is shown.

[0163] Table 6: Effect of sugar type on serotype 3 polysaccharides [Table 6]

[0164] For all sugars used, the solubility limit in DMSO appeared to be reached at 60 times the concentration. With the exception of ethanol, dissolution in DMSO was achieved at carbohydrate mass ratios of 30 or more. Reliable dissolution results were observed when the sugar mass ratio was at least 40 times that of the polysaccharide. Ta.

[0165] Another experiment was performed looking at the use of sugar combinations in activated serotype 3 polysaccharide lyophilized preparations. All arms were formulated at a 40x total carbohydrate ratio, which allows for the D The polysaccharide had a molecular weight of 171 kD, as it consistently dissolved in MSO. All arms were prepared using sucrose alone, with trehalose, or with mannitol. The solution was lyophilized to remove water, and then DMSO was added at ambient temperature with mixing. The polysaccharide was redissolved. Whether it was dissolved well or not was confirmed by visual observation. The results are shown in Table 7. is shown in.

[0166] Table 7: Sugar combinations for polysaccharide solubility in DMSO [Table 7]

[0167] Dissolution in DMSO was achieved for all arms tested.

[0168] Example 5: Streptococcus pneumoniae (S. pneumoniae) using reductive amination in DMSO iae) conjugation of serotypes 15A, 16F, 17F, 20, 24F, and 35B Effect of sodium chloride on The activated polysaccharide was mixed with sucrose at a concentration of 5-10% weight / volume in a polypropylene container for 2-3 min. CRM197 was formulated for lyophilization at 6 mg Ps / mL. It was formulated for lyophilization at 6 mg Pr / mL with volume.

[0169] The formulated Ps and CRM197 solutions were lyophilized separately. The 197 material was redissolved in DMSO. For some arms, 5M sodium chloride The stock solution was used to spike the CRM197 solution before lyophilization or the reconstituted Ps solution. A final concentration of 10-100 mM sodium chloride was achieved during conjugation. The process parameters were kept constant in these experiments.

[0170] The reconstituted Ps solution and CRM197 solution were mixed and blended to obtain serotype-specific polysaccharides and Protein concentration was obtained using sodium cyanoborohydride (1 mole / 1 mole of polysaccharide repeating unit). Borohydride was added and conjugation was allowed to proceed for a serotype-specific period. The reduction with sodium hydroxide and final filtration were carried out as described in Example 2.

[0171] result Pneumonia showing the effect of sodium chloride during conjugation on conjugate properties The experimental results for polysaccharides from Streptococcus pneumoniae serotype 20 are shown in Fig. Streptococcus pneumoniae serotype 16F and The experimental results for 24F and 24C are shown in Table 8.

[0172] As shown in Figure 1 for serotype 20, the sodium chloride concentration at the time of conjugation Increasing the concentration (up to 50 mM) resulted in an increase in conjugate size. As shown in Figure 3, increasing the sodium chloride concentration (up to approximately 25 mM) increased the consumption. As shown in Fig. 1, increasing the sodium chloride concentration (up to about 50 mM) increased the free P species and free Pr species. It decreased.

[0173] Table 8. Streptococcus pneumoniae serotypes 16F and 24F Effect of sodium chloride on conjugate properties [Table 8]

[0174] As shown in Table 8 for serotype 16F, 25 mM sodium chloride was used during conjugation. The inclusion of sodium increased the conjugate size and lysine consumption, but not the free P species and As shown in Table 8 for serotype 24F, conjugation and free Pr decreased. The occasional inclusion of 25 mM sodium chloride increased lysine consumption, but free P and free The isolated Pr decreased.

[0175] Similar results were observed for serotypes 15A and 35B. Inclusion of 25 mM sodium chloride during conjugation ensures Compared to the salt case, the conjugate size and lysine consumption increased, but the free polysaccharide and For serotype 35B, 25 mM chloride was added during conjugation. The inclusion of sodium increases the conjugation time compared to the absence of salt. The cell size and lysine consumption increased (data not shown), but free protein decreased. A little.

[0176] Streptococcus pneumoniae (S. pneumoniae) serum using reductive amination in DMSO Effect of salt type and concentration on the conjugation of 17F Activated serotype 17F polysaccharide was prepared in a polypropylene container with 5% sucrose concentration (wt / v). CRM197 was formulated for lyophilization at 6 mg Ps / mL in a sucrose solution containing 1% by weight of sucrose. The solution was formulated for lyophilization at 6 mg Pr / mL with 0.01 mg Pr / mL per volume.

[0177] The formulated Ps and CRM197 solutions were lyophilized separately. 197 material was redissolved in DMSO. For some arms, concentrated salt solution was added to the redissolved P Spike into the Ps solution or Ps-CRM blend to a final concentration of 1–1% at the time of conjugation. The stock solutions used contained 1M or 5M sodium chloride, 1M or 3M salt. Other process parameters were similar in these experiments. was kept constant throughout the experiment.

[0178] The reconstituted Ps and CRM197 solutions were mixed and blended to obtain a concentration of 2.7 g Ps / mL and 1.8 mg CRM197 / mL. Sodium cyanoborohydride (1 mol / polysaccharide) Repeating unit (1 mole) was added and conjugation was allowed to proceed for 1 hour. After the polymerization reaction, sodium borohydride (2 moles / 1 mole of polysaccharide repeating unit) was added. The batch was added to 150 ml of 10 ... The solution was diluted with M sodium chloride, and then potassium phosphate buffer was added to neutralize the pH.

[0179] The conjugate was then filtered using a 300 kDa NMWC membrane at approximately 4°C with 0.05% ( The solution was dialyzed against 150 mM sodium chloride containing (wt / vol) polysorbate 20. The final conjugate solution was aliquoted and kept at 4°C.

[0180] result The results are shown in Table 9. 2% is the limit of detection. [Table 9]

[0181] Regarding sodium chloride and potassium chloride conditions, the salt concentration during conjugation was increased. This increases the conjugate size and lysine consumption, and reduces free P and free Pr. These effects plateaued at approximately 12.5 mM for both salt types. 25 mM and 50 mM magnesium chloride increased the conjugate activity compared to salt-free conditions. However, the magnesium chloride concentration of 25 mM With an increase from 0.01 to 100 mM, the free polysaccharide and free protein levels increased, and and a reduction in the degree of conjugation (measured by lysine loss and conjugate size) Therefore, magnesium chloride should be kept in the concentration range of 0-50 mM. may be preferable.

[0182] Example 6: Formulation of monovalent conjugates Pneumococcal polysaccharide-CRM197 conjugates were prepared according to the methods described in Examples 2 to 5. The required amount of bulk conjugate needed to achieve the desired concentration of each serotype was calculated using the bulk conjugate. The concentrations of individual serotypes (12F) were calculated based on the volume of each sample and the concentration of the individual bulk polysaccharide concentrates. , 15A, 16F, 17F, 23A, 23B, 24F, 31, and 35B) were mixed with excipients. The mixture was sterile filtered and added to APA under mixing conditions. The concentration was 4 μg / mL (weight / volume PnPs), 20 mM histidine, 150 mM NaCl, 0.2% (wt / vol) PS-20 and 0.250 mg / m in the form of APA L (weight / volume Al) was included.

[0183] Example 7: Monovalent Conjugate New Zealand White Rabbit Immunogenicity Study (15A, 16A) F, 17F, 23A, 23B, 24F, 31, and 35B) The immunogenicity of the monovalent conjugate was evaluated in the New Zealand White Rabbit (NZWR) model. Adult New Zealand White rabbits (NZWR, n=3 / group) were cultured on days 0 and 14. On alternate days (alternating sides), each patient received 0.25 mL of the monovalent conjugate vaccine intramuscularly. The monovalent pneumococcal vaccine was administered intramuscularly (IM). 1 μg PnPs (15A, 16F, 17F, 23A, 23B, 24F, 31, or 35B, each conjugated to CRM197 The mice were administered at the start of the study (before immunization), on day 14 (post-administration 1, PD1), and on day 28 ( Serum was collected on post-dose day 2 (PD2). Animals were monitored by an experienced animal care center for signs of disease or distress. NZWR were observed at least once daily by a medical officer. The study appeared to be safe and well tolerated. Both animal studies were conducted at the National Institutes of Health. All experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Animals. The protocol was developed by Merck & Co., Inc. (Kenilworth, NJ) and and Covance (Denver, PA) Institutional Animal Care and Use Committees Approved by.

[0184] NZWR serum was used for ELI using individual PnPs coating concentrations of 1–2 mg / mL. The IgG immunogenicity was evaluated by SA assay. Functional antibodies were confirmed by immunophagocytosis assay (OPA). -Aguilar et al., 2017, Vaccine 35:865-72 and Burton et al., 2006, Clin Vaccine Immu See nol 13(9):1004-9.

[0185] All monovalent pneumococcal vaccines are immunogenic in rabbits and kill individual bacterial strains. It was found that serotype 12F elicited the development of functional antibodies (data not shown). It was found to be immunogenic in mice (data not shown).

Claims

1. 1. A method for producing a polysaccharide-protein conjugate, the method comprising reacting a polysaccharide in a first solution with a protein in a second solution to form a third solution, and conducting a polysaccharide-protein conjugation reaction in the third solution to produce the polysaccharide-protein conjugate, wherein the third solution comprises at least 1 mM salt; wherein the polysaccharide is from S. pneumoniae serotype 15A, 16F, 17F, 20, 23A, 24F or 35B; wherein the salt is sodium chloride or potassium chloride; wherein said protein is CRM197, and wherein the first solution and the second solution each contain DMSO; method.

2. 2. The method of claim 1, wherein the concentration of sodium chloride in the third solution is 1 to 100 mM.

3. The method described in claim 1, wherein the concentration of potassium chloride in the third solution is 1 to 50 mM.

4. The method of claim 1 , wherein the salt is present in a first solution.

5. The method of claim 1 , wherein the salt is present in the second solution containing the protein.

6. The method of any one of claims 1 to 5, wherein the first solution and the second solution are the same.

7. 10. The method of claim 1, wherein the salt is added so as to be present in the third solution.

8. The method according to any one of claims 1 to 7, wherein the conjugation reaction is a Schiff base reduction or a reductive amination.

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