METHOD TO PRODUCE A GLYCOCJUGATE THAT UNDERSTANDS A SACARIDE CONJUGATED TO A PROTEIN

专利摘要:
abstract "glycoconjugation process" the present disclosure relates in general to methods for preparing glycoconjugates containing a saccharide conjugated to a carrier protein by the use of an agent / oxidant related to a stable nitroxyl radical as an oxidizing agent, to compositions immunogenic substances that comprise these glycoconjugates, and methods for using these glycoconjugates and immunogenic compositions.
公开号:BR112015015031B1
申请号:R112015015031-4
申请日:2013-12-13
公开日:2020-07-14
发明作者:Mingming Han；Rajesh Kumar Kainthan；Jin-Hwan Kim；Avvari Krishna Prasad
申请人:Pfizer Inc；
IPC主号:

专利说明:

FIELD
[001] The present disclosure relates, in general, to methods for preparing glycoconjugates containing a saccharide conjugated to a carrier protein using TEMPO / NCS as an oxidizing agent, to immunogenic compositions comprising those glycoconjugates, and to methods for the use of these glycoconjugates and immunogenic compositions. The present disclosure also relates to methods for preparing glycoconjugates containing a saccharide conjugated to a carrier protein, using stable nitroxyl or nitroxide radicals, such as piperidine-N-oxide or pyrrolidine-N-oxide compounds in the presence of a oxidizer to selectively oxidize primary hydroxyls of said saccharide, to immunogenic compositions comprising these glycoconjugates, and to methods for using these glycoconjugates and immunogenic compositions. FUNDAMENTALS
[002] Polysaccharide protein conjugate vaccines are manufactured using polysaccharides, usually from the surface coating of bacteria, linked to protein carriers. The chemical bonding of the polysaccharide and protein carrier induces an immune response against bacteria that exhibit the polysaccharide contained in the vaccine or on its surface, thus preventing the disease. Correspondingly, vaccination using polysaccharides from pathogenic bacteria is a potential strategy to stimulate host immunity. The polysaccharides that line bacteria vary considerably, even in a single species of bacteria. For example, in Streptococcus pneumoniae (a major cause of meningitis, pneumonia, and serious invasive diseases in babies and young children around the world), there are more than 90 different serotypes due to the variation in the bacterial polysaccharide lining. Therefore, polysaccharide vaccines generally consist of a panel of polysaccharides to increase protection.
[003] Although polysaccharides are immunogenic in themselves, the conjugation of polysaccharides in protein carriers has been used to improve immunogenicity. The carrier protein can be a related protein antigen from the target pathogen, inducing the specific immune response to that pathogen, or a generically immunogenic protein that serves as an adjuvant or general immune response stimulant.
[004] Multivalent pneumococcal polysaccharide-protein conjugate vaccines have been licensed for many years and have proven to be valuable in preventing pneumococcal diseases in babies and have recently been recommended for adults. SUMMARY
[005] In one aspect, the present disclosure provides a method for producing a glycoconjugate comprising a saccharide conjugated to a carrier protein, comprising the steps of: a) reacting a saccharide with 2,2,6,6-tetramethyl-1 - piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups. In an additional aspect, the degree of oxidation of the activated saccharide varies from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 5, from 3 to 40, from 3 to 30, from 3 to 20, from 3 to 10, from 4 to 40, from 4 to 30, from 4 to 20, from 4 to 10, from 5 to 30, from 5 to 25, from 5 to 20, from 5 to 10, from 6 to 50, from 6 to 40, from 6 to 30, from 6 to 20, from 6 to 15, from 6 to 14, from 6 to 13, from 6 to 12, from 6 to 11, from 6 to 10, from 7 to 40, from 7 to 30, from 7 to 20, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12, from 7 to 11, from 7 to 10, from 8 to 40, 8 to 30, 8 to 20, 8 to 15, 8 to 14, 8 to 13, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 40, from 9 to 30, from 9 to 20, from 9 to 15, from 10 to 40, from 10 to 30, from 10 to 20, or from 10 to 15. In an additional aspect, the degree of oxidation of the saccharide activated is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
[006] In a further aspect, the present disclosure provides a method for producing a glycoconjugate comprising a saccharide conjugated to a carrier protein, comprising the steps of: a) reacting a saccharide with a nitroxyl or nitroxide stable radial compound, such as piperidine-N-oxy or pyrrolidine-N-oxy compounds, in the presence of an oxidizer to selectively oxidize primary hydroxyls of said saccharide to produce an activated saccharide containing aldehyde groups; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups.
[007] In the said reaction, the current oxidant is the N-oxoammonium salt, in a catalytic cycle. Preferably, nitroxyl or nitroxide stable radial compounds have the ability to selectively oxidize primary alcohol to aldehydes, in the presence of an oxidant, without over-oxidation to carboxylic acids.
[008] In one aspect, step a) of the reaction is carried out in an aqueous solvent. In another aspect, step a) is carried out in an aprotic solvent. In one aspect, step a) is carried out in a solvent of DMSO (dimethyl sulfoxide), Dimethylacetamide (DMA), Sulfolane, N-Methyl-2-pyrrolidone (NMP), Hexamethylphosphoramide (HMPA) or in DMF (dimethylformamide).
[009] In one aspect, unreacted aldehyde groups are converted back to primary alcohols during a capping step, using borohydride, after conjugation with the carrier protein, thus minimizing the modification of saccharide epitope during the steps of modification involving oxidation followed by conjugation.
[010] In one aspect, said nitroxyl or nitroxide stable radical compounds are composed of piperidine-N-oxy or pyrrolidine-N-oxy. Preferably, said compounds have the ability to oxidize primary alcohols in the presence of an oxidant, to generate aldehyde groups, without affecting secondary hydroxyl groups. More preferably, said compounds have the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without over-oxidation in carboxyl groups.
[011] In one aspect, said nitroxyl or nitroxide stable radical compound bears a TEMPO portion or a PROXIL portion (2,2,5,5-tetramethyl-1-pyrrolidinyloxy). Preferably, said compound has the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without affecting secondary hydroxyl groups. More preferably, said compound has the ability to selectively oxidize primary alcohols in the presence of an oxidant, to generate aldehyde groups, without over-oxidation in carboxyl groups.
[012] In one aspect, said nitroxyl stable radial compound is TEMPO or a derivative thereof. In one aspect, said nitroxyl stable radial compound is selected from the group consisting of TEMPO, 2,2,6,6-Tetramethyl-4- (methyl sulfonyloxy) -1- piperidinooxy, 4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO, 4-lsothiocyanate-TEMPO, 4- (2-lodoacetamido) -TEMPO free radical, 4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO, 4 - (2-Bromoacetamido) -TEMPO, 4-Amino-TEMPO, 4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, said nitroxyl stable radial compound is TEMPO.
[013] In an additional aspect, said nitroxyl stable radial compound is selected from the group consisting of 3p-DOXYL-5a-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, Methyl 5- DOXYL-stearate, 3- (Aminomethyl) -PROXYL, 3-Carbamoyl-PROXYL, 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL, 3-Cyano- PROXIL
[014] In one aspect, said oxidant is a molecule carrying an N-halo portion. Preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of a nitroxyl radical compound.
[015] In one aspect, said oxidant is selected from the group consisting of N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide, Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan- 2,4,6-trione, Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, Diiodoisocyanuric acid and 1,3,5-triiodo-1,3, 5-triazinan-2,4,6-trione. Preferably, said oxidant is N-Chlorosuccinimide.
[016] In one aspect, the degree of oxidation of the activated saccharide ranges from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 10, from 1 to 5, from 3 to 40, 3 to 30, 3 to 20, 3 to 10, 4 to 40, 4 to 30, 4 to 20, 4 to 10, 5 to 30, 5 to 25, 5 to 20, from 5 to 10, from 6 to 50, from 6 to 40, from 6 to 30, from 6 to 20, from 6 to 15, from 6 to 14, from 6 to 13, from 6 to 12, from 6 to 11, from 6 to 10, from 7 to 40, from 7 to 30, from 7 to 20, from 7 to 15, from 7 to 14, from 7 to 13, from 7 to 12, from 7 to 11, from 7 to 10, 8 to 40, 8 to 30, 8 to 20, 8 to 15, 8 to 14, 8 to 13, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 40, 9 to 30, 9 to 20, 9 to 15, 10 to 40, 10 to 30, 10 to 20, or 10 to 15. In an additional aspect, the degree of oxidation activated saccharide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
[017] In one aspect, saccharide is reacted with 0.1 to 10 molar equivalents of oxidant. Preferably, the saccharide is reacted with 0.2 to 5, 0.5 to 2.5 or 0.5 to 1.5 molar equivalents of oxidant. In one aspect, the polysaccharide is reacted with about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2 , 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8 or 5 molar equivalents of oxidant.
[018] In one aspect, the nitroxyl or nitroxide stable radical compound is present in a catalytic amount. In one aspect, saccharide is reacted with less than about 0.3 molar equivalent of a nitroxyl or nitroxide stable radical compound. In one aspect, saccharide is reacted with less than about 0.005 molar equivalent of a nitroxyl or nitroxide stable radical compound. In one aspect, the saccharide is reacted with about 0.005, 0.01, 0.05 or 0.1 molar equivalent of nitroxyl or nitroxide stable radical compound.
[019] In an additional aspect, saccharide is a bacterial capsular polysaccharide. In another aspect, saccharide is a synthetically derived oligo or polysaccharide. In one aspect, the capsular polysaccharide is derived from S. pneumonia (Pn). In an additional aspect, the capsular polysaccharide is selected from capsular polysaccharides of Pn-serotype 10A, Pn-serotype 12F, and Pn-serotype 33F. For example, in one aspect, the capsular polysaccharide is a capsular polysaccharide of Pn-serotype 12F.
[020] In an additional aspect, the capsular polysaccharide is derived from N. meningitidis. In one aspect, the capsular polysaccharide is selected from meningococcal (Mn) -serotype A, C, W135, and Y capsular polysaccharides.
[021] In an additional aspect, the capsular polysaccharide is a meningococcal capsular polysaccharide (Mn) -serotype X.
[022] In an additional aspect, the capsular polysaccharide is derived from Group B Streptococcus (GBS). In one aspect, the capsular polysaccharide is selected from GBS la, lb, II, III, IV, V, VI, VII and VIII serotypes.
[023] In one aspect, the present disclosure provides any of the methods disclosed in this document wherein the carrier protein is a tetanus toxin, diphtheria, whooping cough, Pseudomonas, E. coli, Staphylococcus or Streptococcus. In one aspect the carrier protein is CRM197.
[024] In an additional aspect, the present disclosure provides a method as described in this document, wherein prior to step a), the saccharide is hydrolyzed to a molecular weight ranging from 100 to 400 kDa. For example, in one aspect, the molecular weight ranges from 100 to 350 kDa, from 100 to 300 kDa, from 100 to 250 kDa, from 100 to 200 kDa, from 100 to 150 kDa, from 200 to 400 kDa, from 200 to 350 kDa, from 200 to 300 kDa, from 200 to 250 kDa, from 300 to 400 kDa, or from 300 to 350 kDa.
[025] In a further aspect, the present disclosure provides any of the methods provided herein that further comprise the step of purifying the activated polysaccharide prior to step b). In a further aspect, the methods further comprise the step of adding a reducing agent following step b). In one aspect, the reducing agent is NaCNBHa. In an additional aspect, the methods further comprise the step of adding NaBI-k following the addition of NaCNBHa. In a further aspect, the method comprises a purification step following the addition of NaBI-U.
[026] In another aspect, the present disclosure provides a glycoconjugate produced, or obtainable by any of the methods disclosed in this document. For example, in one aspect, the present disclosure provides a glycoconjugate comprising a saccharide conjugated to a carrier protein that is produced or obtainable by the method comprising the steps of: a) reacting a saccharide with 2,2,6,6-tetramethyl -1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups. In a further aspect, the present disclosure provides a glycoconjugate that comprises a saccharide conjugated to a carrier protein that is produced or obtainable by the method comprising the steps of: a) reacting a saccharide with a nitroxyl or nitroxide stable compound and a oxidizer to produce an activated saccharide containing aldehyde groups; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups. The nitroxyl stable radial and oxidant compounds can be as defined on pages 2 to 4 above.
[027] In a further aspect, the present disclosure provides an immunogenic composition that comprises any of the glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier or diluent. In an additional aspect, the immunogenic composition comprises an additional antigen. In an additional aspect, the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from S. pneumoniae. For example, in one aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from capsular polysaccharides of Pn-serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F, 22F and 23F. In an additional aspect, the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from N. meningitidis. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from serotype capsular polysaccharides A, C, W135 and Y. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide from serotype X capsular polysaccharides. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide derived from Group B Streptococcus (GBS). In one aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from serotypes GBS la, lb, II, III, IV, V, VI, VII and VIII.
[028] In a further aspect, the present disclosure provides any of the immunogenic compositions disclosed herein, which further comprises an adjuvant. In one aspect, the adjuvant is an aluminum-based adjuvant. In an additional aspect, the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide.
[029] In another aspect, the present disclosure provides a method for preventing, treating or alleviating an infection, disease or bacterial condition in an individual, which comprises administering to the individual an immunologically effective amount of any of the immunogenic compositions disclosed herein. In one aspect, the infection, disease or condition is associated with the bacterium S. pneumoniae. In an additional aspect, the infection, disease or condition is associated with the bacterium N. meningitidis.
[030] In another aspect, the present disclosure provides a method for inducing a protective immune response in an individual, which comprises administering to the individual an immunologically effective amount of any of the immunogenic compositions disclosed herein.
[031] In another aspect, the present disclosure provides an immunogenic composition comprising a Pn-serotype 12F conjugated to a carrier protein in which the conjugate is stable. For example, in one aspect, the present disclosure provides an immunogenic composition comprising a Pn-serotype 12F conjugated to a carrier protein, wherein the amount of free Pn-serotype 12F polysaccharide in the composition is less than 35% after 120 days at from when it was prepared. In an additional aspect, the amount of free Pn-serotype 12F polysaccharide is less than 30%, less than 28%, less than 27%, less than 26%, or less than 25% after 120 days from when it was prepared. In an additional aspect, the amount of free Pn-serotype 12F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25% after 90 days from from when it was prepared. In an additional aspect, the amount of free Pn-serotype 12F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25% after 60 days from from when it was prepared. In an additional aspect, the amount of free Pn-serotype 12F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25% after 30 days from from when it was prepared. In a further aspect, the present disclosure provides a composition comprising Pn-serotype 3, 10A, or 33F conjugated to a carrier protein, wherein the amount of free Pn-serotype 3, 10A, or 33F polysaccharide, respectively, in the composition it is less than 35% after 120 days from when it was prepared. In an additional aspect, the amount of free Pn-serotype 3, 10A, or 33F polysaccharide is less than 30%, less than 28%, less than 27%, less than 26%, or less than 25% after 120 days at from when it was prepared. In an additional aspect, the amount of free Pn-serotype 3, 10A, or 33F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25 % after 90 days from when it was prepared. In an additional aspect, the amount of free Pn-serotype 3, 10A, or 33F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25 % after 60 days from when it was prepared. In an additional aspect, the amount of free Pn-serotype 3, 10A, or 33F polysaccharide is less than 35%, less than 30%, less than 28%, less than 27%, less than 26%, or less than 25 % after 30 days from when it was prepared. In one aspect, the amount of free polysaccharide as discussed above is measured at 25 ° C. In one aspect, the carrier protein in the compositions disclosed above consists of a tetanus toxin, diphtheria, whooping cough, Pseudomonas, E. coli, Staphylococcus or Streptococcus. In an additional aspect, the carrier protein is CRM197.
[032] The present disclosure further provides an immunogenic composition comprising any of the glycoconjugates disclosed above and a pharmaceutically acceptable excipient, carrier or diluent. In an additional aspect, these immunogenic compositions comprise an additional antigen. For example, in one aspect, the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from S. pneumoniae. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from capsular polysaccharides of Pn-serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11 A, 14, 15B, 18C, 19A, 19F, 22F, and 23F. In another additional aspect, the additional antigen comprises a protein antigen or a glycoconjugate of a capsular polysaccharide derived from N. meningitidis. In yet another additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from capsular polysaccharides of serotypes A, C, W135 and Y. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide from serotype X capsular polysaccharides. In an additional aspect, the additional antigen comprises a glycoconjugate of a capsular polysaccharide from Group B Streptococcus (GBS). In one aspect, the capsular polysaccharide is selected from GBS la, lb, II, III, IV, V, VI, VII and VIII serotypes.
[033] In a further aspect, these immunogenic compositions also comprise an adjuvant. For example, in one aspect, the adjuvant is an aluminum-based adjuvant. In an additional aspect, the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, and aluminum hydroxide. BRIEF DESCRIPTION OF THE DRAWINGS
[034] Figure 1 shows the structure of the Pn-serotype 12F capsular polysaccharide.
[035] Figure 2 shows the dependence of N-Chlorosuccinimide on the oxidation reaction of Tempo / NCS on the degree of oxidation (DO).
[036] Figure 3 shows the structure of the Pn-serotype 10A capsular polysaccharide.
[037] Figure 4 shows the structure of the capsular polysaccharide of Pn-serotype 33F.
[038] Figure 5 shows the structure of the capsular polysaccharide of Pn-serotype 3.
[039] Figure 6 shows the putative mechanism of oxidation / conjugation of Pn-serotype 12F using TEMPO / NCS.
[040] Figure 7 shows the stability comparison of Pn-serotype 12F conjugates prepared using periodate vs. oxidation. TEMPO / NCS oxidation. DETAILED DESCRIPTION
[041] The present disclosure can be understood more readily with reference to the following detailed description of the various modalities of the disclosure and the examples included in this document. Except where otherwise stated, all technical and scientific terms used in this document have the same meaning as commonly understood by an individual with common knowledge in the technique to which the disclosure belongs. Although any methods and materials similar or equivalent to those described in this document may be used in the practice or testing of the present disclosure, certain preferred methods and materials are described in this document. In describing the modalities and the claims, a particular terminology will be used according to the definitions presented below.
[042] Depending on the usage in question, the singular forms "one", "one", "o", and "a" include references in the plural except where otherwise indicated. Therefore, for example, references to the "method" include one or more methods, and / or steps of the type described in this document and / or that will become apparent to an individual with common knowledge in the art upon reading this disclosure.
[043] Depending on the usage in question, the term “about” means within a statistically significant range of a value, such as a defined concentration range, time duration, molecular weight, temperature or pH. This range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% or within 1% of a given value or range. Occasionally, this range may be within an experimental error typical of standard methods used for measuring and / or determining a given value or range. The allowable variation covered by the term "about" will depend on the particular system under study, and can be readily assessed by an individual with common knowledge in the art. Whenever a banner is cited in this application, any entire number within the banner is also considered a form of disclosure.
[044] It is noted that in this disclosure, terms such as "comprises," "understood," "comprising," "contains," "containing" and the like may have the meaning attributed to them in US patent law; for example, they can mean "includes," "included," "including" and the like. These terms refer to the inclusion of particular ingredients or a set of ingredients without excluding any other ingredients. Terms such as “which essentially consists of” and “consists essentially of” have the meaning given to them in US patent law; for example, they allow the inclusion of additional ingredients or steps that do not detract from the innovative or basic features of the disclosure, that is, they exclude additional ingredients or unmentioned steps that detract from the innovative or basic features of the disclosure. The terms "consists of" and "that consists of" have the meaning given to them in US patent law; that is, that these terms are limited. Correspondingly, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.
[045] Depending on the usage in question, the term "saccharide" can be used to refer to a polysaccharide, an oligosaccharide, or a monosaccharide.
[046] Depending on the usage in question, the term "degree of oxidation" in reference to a saccharide refers to the ratio of the moles of repeating unit of saccharide per mole of aldehyde. The degree of oxidation of a saccharide can be determined using routine methods known to those skilled in the art.
[047] The term "conjugates" or "glycoconjugates" according to the use in question refers to a saccharide covalently conjugated to a carrier protein. The glycoconjugates of the disclosure and the immunogenic compositions that comprise them may contain a certain amount of free saccharide.
[048] The term “free saccharide” according to the use in question means a saccharide that is not covalently conjugated to the carrier protein, but is nevertheless present in the glycoconjugate composition. The free saccharide can be non-covalently associated (i.e., non-covalently bound, absorbed, or trapped) with the conjugated saccharide-carrier protein glycoconjugate. The terms "free polysaccharide" and "free capsular polysaccharide" can be used in this document to convey the same meaning in relation to glycoconjugates in which the saccharide is a polysaccharide or a capsular polysaccharide, respectively.
[049] Depending on the usage in question, "conjugate," "conjugate (a)" and "conjugating" refer to a process by which a saccharide, for example, a bacterial capsular polysaccharide, is covalently attached to a carrier molecule or carrier protein. The conjugation can be carried out according to the methods described below or by other processes known in the art. The conjugation enhances the immunogenicity of the bacterial capsular polysaccharide.
[050] The term "individual" refers to a mammal, including a human being, or to a bird, fish, reptile, amphibian or any other animal. The term "individual" also includes domestic animals or research animals. Non-limiting examples of domestic animals or research animals include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, monkeys, birds, snakes, lizards, fish, turtles, and frogs. The term "individual" also includes farm animals. Non-limiting examples of farm animals include: alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese and turkeys. GLYCOCONJUGATES
[051] The present disclosure relates to methods for preparing glycoconjugates that comprise a saccharide conjugated to a carrier protein, in particular, using a nitroxyl or nitroxide stable radial compound to selectively oxidize primary saccharide alcohols to aldehydes, using , still, an oxidizer. In one aspect, said nitroxyl stable radial compounds are compounds of piperidine-N-oxy or pyrrolidine-N-oxy. Preferably, said compounds have the ability to selectively oxidize primary alcohol to aldehydes in the presence of an oxidant, without over-oxidation to carboxylic acids and without affecting secondary hydroxyl groups. In one aspect, said nitroxyl stable radial compound is a molecule carrying a TEMPO portion or a PROXIL portion (2,2,5,5-tetramethyl-1-pyrrolidinyloxy). Preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without affecting the secondary hydroxyl groups. More preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without over-oxidation in carboxyl groups. In one aspect, said nitroxyl stable radial compound is selected from the groups consisting of TEMPO, 2,2,6,6-Tetramethyl-4- (methyl sulfonyloxy) -1-piperidinooxy, 4-Phosphonooxi- TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO, 4-lsothiocyanate-TEMPO, radial free of 4- (2-lodoacetamido) -TEMPO, 4-Hydroxy-TEMPO, 4- Cyano-TEMPO, 4-Carboxy-TEMPO, 4 - (2-Bromoacetamido) -TEMPO, 4-Amino-TEMPO, 4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, said nitroxyl stable radial compound is TEMPO. In an additional aspect, said nitroxyl stable radial compound is selected from the groups consisting of 30-DOXYL-5a-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, Methyl 5-DOXYL stearate , 3- (Aminomethyl) -PROXYL, 3-Carbamoyl-PROXYL, 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL, 3-Cyano-PROXYL. In one aspect, the oxidant is a molecule carrying an N-halo moiety. Preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of a nitroxyl radical compound. In one aspect, said oxidant is selected from the group consisting of N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide, Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4 , 6-trione, Dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, Diiodoisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinan -2,4,6-trione. Preferably, said oxidant is N-Chlorosuccinimide.
[052] In one aspect, the present disclosure relates to methods for preparing glycoconjugates that comprise a saccharide conjugated to a carrier protein, in particular, using 2,2,6,6-Tetramethyl-1-piperidinyloxy free radical ( TEMPO) to oxidize primary saccharide alcohols to aldehydes using N-Chlorosuccinimide (NCS) as the co-oxidant.
[053] In the glycoconjugates of the disclosure, saccharide is selected from the group consisting of a polysaccharide, an oligosaccharide, and a monosaccharide, and the carrier protein is selected from any suitable carrier as described in this document or known to individuals skilled in the art. In some embodiments, saccharide is a polysaccharide, in particular, a bacterial capsular polysaccharide, such as Streptococcus pneumoniaeserotype 10A (Pn-serotype 10A), Pn-serotype 12F, or Pn-serotype 33F. In some modalities, the carrier protein is CRM197.
[054] Capsular polysaccharides can be prepared by standard techniques known to those skilled in the art. For example, capsular polysaccharides can be prepared from a variety of serotypes, such as 1,3,4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of Streptococcus pneumoniae. These pneumococcal conjugates are prepared by separate processes and formulated in a single dosage formulation. For example, in one embodiment, each pneumococcal polysaccharide serotype is developed in a soy-based medium. The individual polysaccharides are then purified by centrifugation, precipitation, ultrafiltration, and column chromatography. Purified polysaccharides are chemically activated to make saccharides (i.e., activated saccharides) capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. The chemical activation of the polysaccharides and the subsequent conjugation to the carrier protein can be achieved by conventional means. See, for example, U.S. Patents 4,673,574, U.S. 4,902,506, U.S. 7,709,001, and U.S. 7,955,605.
[055] In one embodiment, the glycoconjugate of the disclosure has a molecular weight between about 50 kDa and about 20,000 kDa. In another embodiment, the glycoconjugate has a molecular weight between about 200 kDa and about 10,000 kDa. In another embodiment, the glycoconjugate has a molecular weight between about 500 kDa and about 5,000 kDa. In one embodiment, the glycoconjugate has a molecular weight between about 1,000 kDa and about 3,000 kDa. In other embodiments, the glycoconjugate has a molecular weight between about 600 kDa and about 2,800 kDa; between about 700 kDa and about 2,700 kDa; between about 1,000 kDa and about 2,000 kDa; between about 1,800 kDa and about 2,500 kDa; between about 1,100 kDa and about 2,200 kDa; between about 1,900 kDa and about 2,700 kDa; between about 1,200 kDa and about 2,400 kDa; between about 1,700 kDa and about 2,600 kDa; between about 1,300 kDa and about 2,600 kDa; between about 1,600 kDa and about 3,000 kDa. Any integer within any of the previous ranges is considered a form of disclosure.
[056] Innovative features of the glycoconjugates of the disclosure include the molecular weight profiles of the resulting saccharides and conjugates, the ratio between lysines conjugated to the carrier protein and the number of lysines covalently linked to the polysaccharide, the number of covalent bonds between the carrier protein and the saccharide as a function of saccharide repeat units, and the relative amount of free saccharide compared to total saccharide.
[057] In another embodiment, the polysaccharide is a capsular polysaccharide derived from Neisseria meningitidis. In some embodiments, the capsular polysaccharide is selected from the group consisting of capsular polysaccharides of serotype A, B, C, W135, X and Y of N. meningitidis. In one embodiment, the capsular polysaccharide is a capsular polysaccharide of serotype C. In another embodiment, the capsular polysaccharide is a capsular polysaccharide of serotype W135. In another embodiment, the capsular polysaccharide is a capsular polysaccharide of serotype Y.
[058] In some embodiments, the glycoconjugate of the disclosure comprises a bacterial capsular polysaccharide, in which the capsular polysaccharide has a molecular weight between 10 kDa and 2,000 kDa or between 50 kDa and 1,000 kDa. In some embodiments, the capsular polysaccharide is derived from S. pneumoniae or N. meningitidis. In some embodiments, the capsular polysaccharide is derived from S. pneumoniae is selected from the group consisting of serotype 1.3 capsular polysaccharides , 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In other embodiments, the capsular polysaccharide is derived from N. meningitidise is selected from the group consisting of serotype A, B, C, W135, X and Y capsular polysaccharides.
[059] In one embodiment, the disclosure provides a glycoconjugate that comprises a capsular polysaccharide covalently conjugated to a carrier protein, having one or more of the following resources: the polysaccharide has a molecular weight between 50 kDa and 1,000 kDa; glycoconjugate has a molecular weight between 1,000 kDa to 3,000 KDa; and the conjugate comprises less than about 45% free polysaccharide relative to the total polysaccharide. In some embodiments, the polysaccharide has a molecular weight between 10 kDa and 2,000 kDa. In some embodiments, glycoconjugate has a molecular weight between 50 kDa and 20,000 kDa. In other embodiments, glycoconjugate has a molecular weight between 200 kDa and 10,000 kDa. In other embodiments, the conjugate comprises less than about 30%, 20%, 15%, 10%, or 5% of free polysaccharide relative to the total polysaccharide. The amount of free polysaccharide can be measured as a function of time, for example, after 10, 20, 30, 40, 50, 60, 70, 80, 90, or 120 days, or even more, after the conjugate has been prepared .
[060] The number of lysine residues in the saccharide-conjugated carrier protein can be characterized as a range of conjugated lysines, which can be expressed as a molar ratio. For example, in an immunogenic composition where 4 to 15 lysine residues of CRM197 are covalently bound to the saccharide, the molar ratio between conjugated lysines and CRM197 in the glycoconjugate is between about 10: 1 and about 40: 1. In an immunogenic composition where 2 to 20 lysine residues of CRM197 are covalently bound to the saccharide, the molar ratio between conjugated lysines and CRM197 in the glycoconjugate is between about 5: 1 and about 50: 1.
[061] In one embodiment, the molar ratio between conjugated lysines and carrier protein is about 10: 1 to about 25: 1. In some modalities, the carrier protein is CRM197.
[062] In one modality, the saccharide: carrier protein (w / w) ratio is between 0.2 and 4. In another modality, the saccharide: carrier protein (w / w) ratio is between 1.1 and 1.7 . In some modalities, saccharide is a bacterial capsular polysaccharide, and the saccharide: carrier protein (w / w) ratio is between 0.2 and 4. In other modalities, saccharide is a bacterial capsular polysaccharide, and the saccharide: protein ratio carrier (w / w) is between 1.1 and 1.7. In some modalities, the carrier protein is CRM197.
[063] The frequency of attachment of the saccharide chain to a lysine in the carrier protein is another parameter that serves to characterize the glycoconjugates of the staining. For example, in one embodiment, there is at least one covalent bond between the carrier protein and the polysaccharide for every 100 polysaccharide repeat units. In one embodiment, there is at least one covalent bond between the carrier protein and the polysaccharide for every 50 repeat units of the polysaccharide saccharide. In one embodiment, there is at least one covalent bond between the carrier protein and the polysaccharide for every 25 repeat units of the polysaccharide saccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 4 repeat units of the polysaccharide saccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 10 repeat units of the polysaccharide saccharide. In an additional embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 15 repeat units of the polysaccharide saccharide.
[064] In frequent modalities, the carrier protein is CRM 197 and the covalent link between CRMigze and the polysaccharide occurs at least once every 4, 10, 15 or 25 repeat units of polysaccharide saccharide. In some embodiments, the polysaccharide is a bacterial capsular polysaccharide, for example, a capsular polysaccharide derived from S. pneumoniae or N. meningitidis bacteria.
[065] In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for each 5 to 10 saccharide repeat units; every 2 to 7 saccharide repeat units; every 3 to 8 saccharide repeat units; every 4 to 9 saccharide repeat units; each 6 to 11 saccharide repeat units; each 7 to 12 saccharide repeat units; every 8 to 13 saccharide repeat units; each 9 to 14 saccharide repeat units; every 10 to 15 saccharide repeat units; each 2 to 6 saccharide repeat units, each 3 to 7 saccharide repeat units; every 4 to 8 saccharide repeat units; every 6 to 10 saccharide repeat units; each 7 to 11 saccharide repeat units; each 8 to 12 saccharide repeat units; each 9 to 13 saccharide repeat units; every 10 to 14 saccharide repeat units; every 10 to 20 saccharide repeat units; or every 4 to 25 saccharide repeat units.
[066] In another embodiment, at least one link between the carrier protein and the saccharide occurs for every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24 or 25 polysaccharide saccharide repeat units.
[067] In one embodiment, the glycoconjugate of the disclosure comprises at least one covalent bond between the carrier protein and the polysaccharide for every 25 saccharide repeat units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 4 repeat units of the polysaccharide saccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 10 repeat units of the polysaccharide saccharide. In an additional embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once every 15 repeat units of the polysaccharide saccharide.
[068] In one embodiment, glycoconjugate comprises less than about 45% free saccharide compared to the total amount of saccharide. In another embodiment, the glycoconjugate comprises less than about 30% free saccharide compared to the total amount of saccharide. In another embodiment, the glycoconjugate comprises less than about 20% free saccharide compared to the total amount of saccharide. In an additional embodiment, the glycoconjugate comprises less than about 10% free saccharide compared to the total amount of saccharide. In another embodiment, the glycoconjugate comprises less than about 5% free saccharide compared to the total amount of saccharide.
[069] In another embodiment, glycoconjugate comprises less than about 20 mole% of carrier protein residues compared to the total amount of glycoconjugate.
[070] In another aspect, the disclosure provides an immunogenic composition that comprises a glycoconjugate of the disclosure and at least one among an adjuvant, diluent or carrier.
[071] In one embodiment, the disclosure provides an immunogenic composition comprising a glycoconjugate of the disclosure and at least one of the adjuvant, diluent or carrier, wherein the glycoconjugate comprises a bacterial capsular polysaccharide covalently conjugated to a carrier protein. In some embodiments, the capsular polysaccharide is derived from S. pneumoniae or N. meningitidis.
[072] In some embodiments, the immunogenic composition comprises an adjuvant. In some embodiments, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment, the immunogenic composition comprises the aluminum phosphate adjuvant.
[073] In some embodiments, the glycoconjugates or immunogenic compositions of the disclosure can be used to generate antibodies that are functional as measured by killing bacteria in an animal efficacy model or through an opsonophagocytic extermination assay.
[074] In one embodiment, the disclosure provides a method for inducing an immune response in an individual, which comprises administering to the individual an immunologically effective amount of an immunogenic composition of the disclosure as described herein. In another aspect, the disclosure provides a method for inducing an immune response against a pathogenic bacterium in an individual, which comprises administering to the individual an immunologically effective amount of an immunogenic composition as described herein. In another aspect, the disclosure provides a method for preventing or alleviating a disease or condition caused by a pathogenic bacterium in an individual, which comprises administering to the individual an immunologically effective amount of an immunogenic composition as described herein. In another aspect, the disclosure provides a method for reducing the severity of at least one symptom of a disease or condition caused by infection with a pathogenic bacterium in an individual, which comprises administering to the individual an immunologically effective amount of an immunogenic composition as described in this document. In some embodiments, the pathogenic bacterium is S. pneumoniae or N. meningitidis.
[075] In addition, the present disclosure provides methods for inducing an immune response against S. pneumoniaeor N. meningitidis bacteria, methods for preventing a disease caused by S. pneumoniaeor N. meningitidis bacteria, and methods for reducing the severity of at least one symptom of a disease caused by infection with bacteria S. pneumoniae or N. meningitidis. SACARIDES
[076] Saccharides include polysaccharides, oligosaccharides and monosaccharides. In some embodiments, saccharide is a polysaccharide, in particular, a bacterial capsular polysaccharide.
[077] The molecular weight of the capsular polysaccharide is a factor for use in immunogenic compositions. Capsular polysaccharides with high molecular weight are capable of inducing certain antibody immune responses due to a greater valence of epitopes present on the antigenic surface. The isolation and purification of capsular polysaccharides with high molecular weight are contemplated for use in the conjugates, compositions and methods of the present disclosure.
[078] In one embodiment, the capsular polysaccharide has a molecular weight between 10 kDa and 2,000 kDa. In one embodiment, the capsular polysaccharide has a molecular weight between 50 kDa and 1,000 kDa. In another embodiment, the capsular polysaccharide has a molecular weight between 50 kDa and 300 kDa. In another embodiment, the capsular polysaccharide has a molecular weight between 70 kDa and 300 kDa. In additional embodiments, the capsular polysaccharide has a molecular weight between 90 kDa and 250 kDa; 90 kDa and 150 kDa; 90 kDa and 120 kDa; 80 kDa and 120 kDa; 70 kDa and 100 kDa; 70 kDa and 110 kDa; 70 kDa and 120 kDa; 70 kDa and 130 kDa; 70 kDa and 140 kDa; 70 kDa and 150 kDa; 70 kDa and 160 kDa; 80 kDa and 110 kDa; 80 kDa and 120 kDa; 80 kDa and 130 kDa; 80 kDa and 140 kDa; 80 kDa and 150 kDa; 80 kDa and 160 kDa; 90 kDa and 110 kDa; 90 kDa and 120 kDa; 90 kDa and 130 kDa; 90 kDa and 140 kDa; 90 kDa and 150 kDa; 90 kDa and 160 kDa; 100 kDa and 120 kDa; 100 kDa and 130 kDa; 100 kDa and 140 kDa; 100 kDa and 150 kDa; 100 kDa and 160 kDa; and similar desired molecular weight ranges. Any integer within any of the previous ranges is considered a form of disclosure.
[079] The S. pneumoniae capsular polysaccharide, Serotype 12F (Pn-serotype 12F) has the structure shown in Figure 1. The S. pneumoniae capsular polysaccharide, Serotype 10A (Pn-serotype 10A) has the structure shown in Figure 3 The S. pneumoniae capsular polysaccharide, Serotype 33F (Pn-serotype 33F) has the structure shown in Figure 4. The S. pneumoniae capsular polysaccharide, Serotype 3 (Pn-serotype 3) has the structure shown in Figure 5.
[080] In some embodiments, capsular polysaccharides, glycoconjugates or immunogenic compositions of the disclosure are used to generate antibodies that are functional as measured by the killing of bacteria in an animal efficacy model or an opsonophagocytic killing assay that demonstrates that antibodies kill off antibodies. bacteria. Capsular polysaccharides can be obtained directly from bacteria using isolation procedures known to individuals with common knowledge in the art. See, for example, Fournier et al. (1984), supra; Fournier et al. (1987) Ann. Inst. Pasteur / Microbiol. 138: 561-567; Patent Application Publication No. 2007/0141077; and International Patent Application Publication No. WO 00/56357; each of which is incorporated by reference as if presented in its entirety). In addition, they can be produced using synthetic protocols. Furthermore, the capsular polysaccharide can be recombinantly produced using genetic engineering procedures also known to an individual with common knowledge in the art (see, Sau et al. (1997) Microbiology143: 2395-2405; and US Patent No. 6,027,925; each of which is incorporated by reference as if presented in its entirety). Strains of S. pneumoniae or N. menigitidis can be used to produce the respective polysaccharides that are obtained either from established culture collections or from clinical specimens. CARRYING PROTEINS
[081] Another component of the disclosure glycoconjugate is a carrier protein to which the saccharide is attached. The term "protein carrier" or "carrier protein" or "carrier" refers to any protein molecule that can be conjugated to an antigen (such as a capsular polysaccharide) against which an immune response is desired.
[082] Conjugation to a carrier can enhance the immunogenicity of the antigen. Protein carriers for the antigens can be toxins, toxoids or any mutant cross-reactive (CRM) material of tetanus toxin, diphtheria, whooping cough, Pseudomonas, E. coll, Staphylococcuse Streptococcus. In one embodiment, a carrier is from the CRM197 diphtheria toxoid, derived from the C. diphtheriae C7 (0197) strain, which produces the CRM197 protein. This strain has accession number ATCC 53281. A method for producing CRM197 is described in U.S. Patent No. 5,614,382, each of which is incorporated by reference as if presented in its entirety. Alternatively, a fragment or epitope of the protein carrier or another immunogenic protein can be used. For example, a haptenic antigen can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (for example, as described in International Patent Application No. WO 2004/083251), E. coliLT, E. coll ST, and exotoxin A from Pseudomonas aeruginosa. use bacterial outer membrane proteins, such as outer membrane complex c (OMPC), porin, transferrin agglutination proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA) or protein D Haemophilus influenzae. proteins such as ovalbumin, californian keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD) can also be used as carrier proteins.
[083] As previously discussed in this document, the number of lysine residues in the carrier protein that become conjugated to the saccharide can be characterized as a range of conjugated lysines. For example, in a given immunogenic composition, CRM197 can comprise from 1 to 15 lysine residues out of 39 covalently linked to saccharide. Another way of expressing this parameter is that about 2.5% to about 40% of CRMi97 lysines are covalently linked to saccharide. For example, in a given immunogenic composition, CRM197 can comprise from 1 to 20 lysine residues among the 39 covalently linked to saccharide. Another way of expressing this parameter is that about 2.5% to about 50% of CRPvW lysines are covalently linked to saccharide. METHODS TO PRODUCE GLYCOCONJUGATES
[084] In order to produce a glycoconjugate, a polysaccharide must first be activated (ie chemically modified) before it can be chemically linked to a carrier, such as a protein. Prior to the activation step, saccharides can be hydrolyzed or mechanically sized by pressure homogenization to achieve appropriate molecular weights (for example, 50 kDa to 500 kDa) for subsequent activation and conjugation. The partial oxidation of carbohydrates to polysaccharides was effectively used to generate aldehyde groups that are then coupled to amine groups, such as the lysine residues of carrier proteins, to generate immunogenic conjugates. It is important that the method used to conjugate a polysaccharide to a carrier protein results in a stable covalent bond, and the reaction conditions are mild enough to maintain the structural integrity of the individual components. Commonly used methods to activate and couple polysaccharides to carrier proteins include reductive amination chemistry (RAC), cyanylation, and use of carbodiimide. Typically, reductive amination involves the use of sodium or potassium periodate or periodic acid in order to selectively oxidize vicinal -OH groups in active aldehyde groups. Cyanylation is used to randomly convert -OH groups to active -CN groups. Carbodiimide is used to activate carboxyl groups by replacing -OH groups with carbodiimide.
[085] Reductive amination chemistry (RAC) consists of one of the most common methods used to couple polysaccharides to proteins since the reaction between the carbonyl group resulting from the polysaccharide and the amino group of the carrier protein can form a corresponding Schiffs base, which it can then be selectively reduced in the presence of sodium cyanoborohydride (NaCNBHs) to a very stable saturable carbon-nitrogen bond. In addition, reductive amination can be carried out in an aqueous solution under conditions mild enough to preserve the structural integrity of the saccharide and protein components. After conjugation, unreacted aldehydes can then be capped by reducing sodium borohydride (NaBH4). The conjugate can then be purified (for example, by ultrafiltration / diafiltration), providing a final bulk glycoconjugate in a succinate buffered saline solution.
[086] However, depending on the particular polysaccharide, the use of the common methods noted above does not always provide adequate results. For example, direct oxidation of polysaccharides with sodium periodate can result in cleavage of the polysaccharide backbone.
[087] For example, it was observed that for conjugates prepared using standard periodate oxidation conditions (followed by reductive amination), representative batches showed an increase in free polysaccharide and a reduction in molecular weight at 25 ° C and above. The present disclosure provides the observation that the use of an oxidation method based on N-oxoammonium salt has resulted in an improved stability of several polysaccharide conjugates of S. pneumoniae, particularly Serotype 12F. In particular, as shown in greater detail in Examples 1 to 7, 2,2,6,6, free radical -Tetramethyl-1-piperidinyloxy (TEMPO) was used in combination with N-chlorosuccinimide (NCS) to effectively oxidize the groups primary hydroxyl serotypes 12F, 10A, 3 and 33F in order to improve the stability of the resulting conjugates. Although the selective oxidation of primary alcohols to aldehydes using TEMPO / NCS has been shown in the context of organic chemical reactions using small molecules in organic solvents (see, for example, Einhorn et al., J. Org. Chem. 61, pp. 7452-7454 (1996)), the present disclosure provides the innovative finding that TEMPO / NCS can be used as an oxidizing agent to selectively oxidize complex polysaccharides in aqueous solution to produce stable polysaccharide protein conjugates.
[088] Correspondingly, in one embodiment, the present disclosure provides methods for producing glycoconjugates that comprise a saccharide conjugated to a carrier protein, comprising the steps of: a) reacting a saccharide with a nitroxyl stable radial compound and a oxidizer to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups.
[089] In one aspect, unreacted aldehyde groups are converted back to primary alcohols during a capping step, using borohydride, after conjugation with the carrier protein, thus minimizing the modification of saccharide epitope during the steps of modification involving oxidation followed by conjugation
[090] In one aspect, step a) of the reaction is carried out in an aqueous solvent. In another aspect, step a) is carried out in an aprotic solvent. In one aspect, step a) is carried out in a solvent of DMSO (dimethyl sulfoxide), Dimethylacetamide (DMA), Sulfolane, N-Methyl-2-pyrrolidone (NMP), Hexamethylphosphoramide (HMPA) or in DMF (dimethylformamide).
[091] In one aspect, said nitroxyl stable radial compounds are composed of piperidine-N-oxy or pyrrolidine-N-oxy. Preferably, said compounds have the ability to selectively oxidize primary alcohols in the presence of an oxidant, to generate aldehyde groups, without affecting the secondary hydroxyl groups. More preferably, said compounds have the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without over-oxidation in carboxyl groups. In one embodiment, said nitroxyl stable radial compound is a molecule carrying a portion of TEMPO or a portion of PROXIL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy). Preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of an oxidant, to generate aldehyde groups, without affecting the secondary hydroxyl groups. More preferably, said molecule has the ability to selectively oxidize primary alcohols in the presence of an oxidant, to generate aldehyde groups, without over-oxidation in carboxyl groups. In one aspect, said nitroxyl stable radial compound is TEMPO or a derivative thereof. In one embodiment, said nitroxyl stable radial compound is selected from the groups consisting of TEMPO, 2,2,6,6-Tetramethyl-4- (methyl sulfonyloxy) -1-piperidinooxy, 4-Phosphonooxy-TEMPO, 4-Oxo-TEMPO, 4-Methoxy-TEMPO, 4-lsothiocyanate-TEMPO, free radical of 4- (2-lodoacetamido) -TEMPO, 4-Hydroxy-TEMPO, 4-Cyano-TEMPO, 4-Carboxy-TEMPO, 4 - (2-Bromoacetamido) -TEMPO, 4-Amino-TEMPO, 4-Acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl. Preferably, said nitroxyl stable radial compound is TEMPO. In an additional embodiment, said nitroxyl stable radial compound is selected from the groups consisting of 30-DOXYL-5a-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, Methyl 5-DOXYL stearate , 3- (Aminomethyl) -PROXYL, 3-Carbamoyl-PROXYL, 3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl, 3-Carboxy-PROXYL, 3-Cyano-PROXYL in one modality, said oxidant is a molecule carrying an N-halo moiety. Preferably, said molecule has the ability to selectively oxidize primary alcohol in the presence of a nitroxyl radical compound. In one embodiment, said oxidant is selected from the group consisting of N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide, Dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4 , 6-trione, Dibromoisocyanuric acid, 1,3,5-tribromo- 1,3,5-triazinan-2,4,6-trione, Diiodoisocyanuric acid and 1,3,5-triiodo- 1,3,5-triazinan -2,4,6-trione. Preferably, said oxidant is N-Chlorosuccinimide.
[092] In one aspect, the saccharide is reacted with 0.1 to 10 molar equivalents of oxidant. Preferably, the saccharide is reacted with 0.2 to 5, 0.5 to 2.5 or 0.5 to 1.5 molar equivalents of oxidant. In one aspect, the polysaccharide is reacted with about 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2 , 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8 or 5 molar equivalents of oxidant.
[093] In one aspect, the nitroxyl stable radial compound is present in a catalytic amount. In one aspect, saccharide is reacted with less than about 0.3 molar equivalent of stable nitroxyl radial compound. In one aspect, saccharide is reacted with less than about 0.005 molar equivalent of stable nitroxyl radial compound. In one aspect, the saccharide is reacted with about 0.005, 0.01, 0.05 or 0.1 molar equivalent of nitroxyl stable radial compound.
[094] In one embodiment, the present disclosure provides methods for producing glycoconjugates that comprise a saccharide conjugated to a carrier protein, comprising the steps of: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amino groups.
[095] In other modalities, the method also comprises a step of purifying the glycoconjugate, for example, by diafiltration.
[096] In each case, the saccharide is selected from the group consisting of a polysaccharide, an oligosaccharide and a monosaccharide.
[097] In each case, said saccharide can be purified from the fermentation medium or synthetically derived.
[098] In frequent modalities, the carrier protein is CRM 197. In one embodiment, the bacterial capsular polysaccharide is a capsular polysaccharide derived from S. pneumoniae. In another preferred embodiment, the bacterial capsular polysaccharide is a capsular polysaccharide derived from N. meningitides.
[099] In one embodiment, the method for producing a glycoconjugate from the disclosure comprises the step of isolating the saccharide-carrier protein conjugate after it has been produced. In frequent modalities, glycoconjugate is isolated by ultrafiltration.
[0100] In one embodiment, the carrier protein used in the method to produce an isolated conjugate of capsular polysaccharide-carrier protein of S. pneumonia comprises CRM197. In one embodiment, the carrier protein used in the method to produce an isolated conjugate of capsular polysaccharide-carrier protein of N. meningitidis comprises CRM197.
[0101] In one embodiment, CRM197 is reacted with the activated polysaccharide in a weight ratio of about 1: 1.
[0102] In one embodiment, the method for producing an isolated conjugate of capsular polysaccharide-carrier protein from S. pneumonia comprises the step of capping the polysaccharide-carrier protein reaction mixture to remove unreacted activation groups.
[0103] In one embodiment, CRM197 in the method for producing a capsular polysaccharide-CRM-197 conjugate is added in a weight ratio of about 0.4: 1 of CRM ^ capsular polysaccharide imolecule. In other embodiments, the weight ratio of CRMi97: capsular polysaccharide is about 0.5: 1, about 0.6: 1, about 0.7: 1, about 0.8: 1, about 0, 9: 1, about 1: 1, about 1.1: 1, about 1.2: 1, about 1.3: 1, about 1.4: 1, or about 1.5: 1 .
[0104] In one embodiment, the saccharide used in the method to produce the glycoconjugate of the disclosure has a molecular weight between about 10 kDa and about 2,000 kDa. In other embodiments, the molecular weight is between about 50 kDa and about 1,000 kDa, between about 50 kDa and about 20,000 kDa, between about 200 kDa and about 10,000 kDa, between about 1,000 kDa and about 3,000 kDa.
[0105] In another aspect, the disclosure provides an immunogenic composition that comprises a glycoconjugate produced by any of the methods described herein.
[0106] In another aspect, the disclosure provides an immunogenic composition comprising a glycoconjugate obtainable by any of the methods described in this document. IMMUNOGENIC COMPOSITIONS
[0107] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen, for example, a microorganism or a component thereof, the composition of which can be used to elicit an immune response in an individual.
[0108] Depending on the use in question, "immunogenic (a)" means the capacity of an antigen (or an epitope of the antigen), such as a bacterial capsular polysaccharide, or a glycoconjugate or immunogenic composition that comprises the antigen, to cause an immune response in a host, such as a mammal, humorally or cellularly mediated, or both.
[0109] Correspondingly, a "glycoconjugate" or "conjugate" according to the use in question means any glycoconjugate containing an antigen or antigenic determinant (ie epitope) from a bacterial capsular polysaccharide conjugate to a carrier molecule that can be used to elicit an immune response.
[0110] Glycoconjugate can serve to sensitize the host by presenting the antigen in association with MHC molecules on a cell surface. In addition, antigen-specific T cells or antibodies can be generated to allow for future protection of an immunized host. Therefore, glycoconjugates can protect the host against one or more symptoms associated with an infection by the bacteria, or they can protect the host from death due to infection with the bacteria associated with the capsular polysaccharide. Glycoconjugates can also be used to generate polyclonal or monoclonal antibodies, which can be used to confer passive immunity on an individual. Glycoconjugates can also be used to generate antibodies that are functional as measured by killing bacteria in an animal efficacy model or through an opsonophagocytic extermination assay.
[0111] An “antibody” is an immunoglobulin molecule capable of specific agglutination to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. Depending on the usage in question, except where otherwise indicated by the context, the term is intended to cover not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (for example, chimeric, humanized and / or derivatives to alter effector functions, stability and other biological activities) and fragments thereof (such as Fab, Fab ', F (ab') 2, Fv), single chain antibodies (ScFv) and domain, including shark and camelid antibodies), and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (for example, bispecific antibodies as long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified configuration of the immunoglobulin molecule that comprises a recognition site antigen. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, lgG1, lgG2, lgG3, lgG4, lgA1 and lgA2 in humans. The heavy chain constant domains that correspond to different classes of immunoglobulins are referred to as alpha, delta, epsilon, gamma, and mu, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are notorious.
[0112] "Antibody fragments" comprise only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all of the functions normally associated with that portion when present in an intact antibody.
[0113] The term "antigen" generally refers to a biological molecule, usually a protein, peptide, polysaccharide or conjugate in an immunogenic composition, or immunogenic substance that can stimulate the production of antibodies or T cell responses, or both in an animal, including compositions that are injected or absorbed into an animal. The immune response can be generated to the whole molecule, or to several portions of the molecule (for example, an epitope or hapten). The term can be used to refer to an individual molecule or to a homogeneous or heterogeneous population of antigenic molecules. An antigen is recognized by antibodies, T cell receptors or other elements of specific humoral and / or cellular immunity. "Antigen" also includes all related antigenic epitopes. Epitopes of a given antigen can be identified using any number of epitope mapping techniques, known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, NJ For example, linear epitopes can be determined, for example, by synthesizing simultaneously large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and the peptides are reacted with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Know. USA 81: 3998-4002; Geysen et al. (1986) Molec. Immunol.23: 709-715; each of which is incorporated by reference as if it were presented in its entirety. Similarly, conformational epitopes can be identified by determining spatial conformation of amino acids, such as, for example, through x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra.
[0114] Additionally, for the purposes of the present disclosure, "antigen" can also be used to refer to a protein that includes modifications, such as deletions, additions and substitutions (generally conservative in nature, but may be non-conservative), to native sequence, as long as the protein maintains the ability to elicit an immune response. These modifications may be deliberate, as through site-directed mutagenesis, or through particular synthetic procedures, or through a genetic engineering approach, or they may be accidental, such as through host mutations, that produce the antigens. In addition, the antigen can be derived, obtained, or isolated from a microbe, for example, a bacterium, or it can be an entire organism. Similarly, an oligonucleotide or polynucleotide, which expresses an antigen, such as in nucleic acid immunization applications, is also included in the definition. Synthetic antigens can also be included, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens (Bergmann et al. (1993) Eur. J. Immunol.23: 2777 2781; Bergmann et al. (1996) J Immunol.157: 3242-3249; Suhrbier (1997) Immunol. Cell Biol.75: 402 408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, from June 28 to July 3, 1998 ).
[0115] A "protective" immune response refers to the ability of an immunogenic composition to elicit an immune response, whether humoral or cell-mediated, or both, which serves to protect an individual against infection. The protection provided does not need to be absolute, that is, the infection does not need to be completely prevented or eradicated. If there is a statistically significant improvement compared to a control population of individuals, for example, infected animals not administered with vaccine or immunogenic composition. Protection may be limited to mitigating the severity or rapidity of the onset of symptoms of infection. In general, a “protective immune response” would include inducing an increase in antibody levels specific to a particular antigen in at least 50% of individuals, including a certain level of measurable functional antibody responses to each antigen. In particular situations, a “protective immune response” could include inducing a double increase in antibody levels or a quadruple increase in antibody levels specific to a particular antigen in at least 50% of individuals, including a certain level of antibody response measurable to each antigen. In certain embodiments, antibody opsonization correlates with a protective immune response. Therefore, a protective immune response can be tested by measuring the percentage reduction in bacterial count in an opsonophagocytosis assay, for example, those described below. Preferably, there is a reduction in bacterial count of at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more. The "immunogenic amount" of a particular conjugate in a composition is generally dosed based on the total, conjugated and unconjugated polysaccharide for that conjugate. For example, a capsular polysaccharide conjugate with 20% free polysaccharide will have about 80 mcg of conjugated polysaccharide and about 20 mcg of unconjugated polysaccharide at a dose of 100 mcg. The protein contribution to the conjugate is generally not considered when calculating the dose of a conjugate. In general, each dose will comprise 0.1 to 100 mcg of polysaccharide, particularly 0.1 to 10 mcg, and, more particularly, 1 to 10 mcg.
[0116] One embodiment of the disclosure provides an immunogenic composition comprising any of the glycoconjugates comprising a S. pneumonia capsular polysaccharide conjugated to a carrier protein described above.
[0117] The immunogenic compositions of the present disclosure can be used to protect or treat a human subject susceptible to bacterial infection, for example, by an S. pneumoniae bacterium or by a N. meningitidis bacterium, through the administration of immunogenic compositions via a systemic route , dermal or mucosal, or can be used to generate a polyclonal or monoclonal antibody preparation that could be used to confer passive immunity in another individual. Such administrations may include injection through intramuscular, intraperitoneal, intradermal or subcutaneous routes; or through mucosal administration to the oral / alimentary, respiratory or genitourinary tracts. Immunogenic compositions can also be used to generate antibodies that are functional as measured by killing bacteria in an animal efficacy model or through an opsonophagocytic killing assay.
[0118] Optimal amounts of components for a particular immunogenic composition can be confirmed by standard studies involving the observation of appropriate immune responses in individuals. After an initial vaccination, individuals may receive one or more booster immunizations that are adequately spaced.
[0119] In one embodiment, the immunogenic compositions of the disclosure further comprise at least one among an adjuvant, a buffer, a cryoprotectant, a salt, a divalent cation, a non-ionic detergent, a free radical oxidation inhibitor, a thinner or a carrier. In one embodiment, the adjuvant in the immunogenic composition of the disclosure is an aluminum-based adjuvant. In one embodiment, the adjuvant is an aluminum-based adjuvant selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide. In one embodiment, the adjuvant is aluminum phosphate.
[0120] An adjuvant is a substance that enhances the immune response when administered with an immunogen or antigen. A number of cytokines or lymphokines have been shown to have immune modulating activity, and therefore may be useful in the same or similar ways to adjuvants, including, without limitation, interleukins 1-a, 1-3, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, US Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and their mutant forms); interferons-a, 0 and Y; macrophage granulocyte colony stimulating factor (GM-CSF) (see, for example, U.S. Patent No. 5,078,996 and ATCC Accession Number 39900); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); and tumor necrosis factors a and 0. Still other adjuvants that are useful with the immunogenic compositions described herein include chemokines, including, without limitation, MCP-1, MIP-1a, MIP-10, and RANTES; adhesion molecules, such as a selectin, for example, L-selectin, P-selectin and E-selectin; mucin-like molecules, for example, CD34, GlyCAM-1 and MadCAM-1; a member of the integrin family, such as LFA-1, VLA-1, Mac-1 and p150.95; a member of the immunoglobulin superfamily, such as PECAM, ICAMs, for example, ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; co-stimulating molecules, such as B7-1, B7-2, CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1, and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6; and Caspases, including ICE.
[0121] Suitable adjuvants used to enhance an immune response may further include, without limitation, MPL ™ (3-O-deacylated monophosphoryl lipid A, Corixa; Hamilton, MT, USA), which is described in US Patent No. 4,912. 094. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa, and those described in US Patent 6,113,918 . An AGP is 2- [(R) -3-Tetradecanoyloxy tetradecanoylamino] ethyl 2-Deoxy-4-O-phosphono-3- O - [(R) -3-tetradecanoyloxy tetradecanoyl] -2 - [(R) -3- tetradecanoyloxy tetradecanoyl-amino] -bD-glycopyranoside, which is also known as 529 (previously known as RC529). This 529 adjuvant is formulated as an aqueous form (AF) or a stable emulsion (SE).
[0122] Still other adjuvants include muramyl peptides, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2- (1 '-2'dipalmitoil -sn-glycero-3-hydroxy phosphoryloxy) -ethylamine (MTP-PE); oil-in-water emulsions, such as MF59 (US Patent No. 6,299,884) (containing 5% Squalene, 0.5% polysorbate 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated in submicron particles using a microfluidizer, such as a Model 110Y microfluidizer (Microfluidics, Newton, MA, USA)), and SAF (containing 10% Squalene, 0.4% polysorbate 80, 5% L121 pluronic-blocked polymer, and thr-MDP, either microfluidized in a submicron emulsion or mixed by vortex to generate an emulsion with larger particle size); incomplete Freund's adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; Amphigenic; Avridine; L121 / equalene; D-lactide-polylactide / glycoside; pluronic polyols; Bordetella exterminated; saponins, such as STIMULON ™ QS-21 (Antigenics, Framingham, MA, USA), described in US Patent No. 5,057,540, ISCOMATRIX ™ (CSL Limited, Parkville, Australia), described in US Patent No. 5,254,339, and immunostimulating complexes (ISCOMS); Mycobacterium tuberculosis; bacterial lipopolysaccharides; synthetic polynucleotides, such as oligonucleotides containing a CpG motif (for example, U.S. Patent No. 6,207,646); IC-31 (Intercell AG, Vienna, Austria), described in the patents NR EP 1,296,713 and EP 1,326,634; a pertussis toxin (PT) or mutant thereof, a cholera toxin or mutant thereof (for example, U.S. Patent Nos. 7,285,281, 7,332,174, 7,361,355 and 7,384,640); or an E. coli thermo labile toxin (LT) or mutant thereof, particularly LT-K63, LT-R72 (for example, U.S. Patent Nos. 6,149,919, U.S. 7,115,730 and U.S. 7,291,588).
[0123] Optionally, the immunogenic composition can comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include carriers approved by a regulatory agency of a federal or state government, or other regulatory agency, or listed in the North American Pharmacopoeia or other pharmacopoeia generally recognized for use on animals, including humans as well as non-human mammals . The term carrier can be used to refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Water, saline solutions and aqueous dextrose and glycerol solutions can be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation must adapt to the mode of administration.
[0124] The immunogenic compositions of the present disclosure may further comprise one or more additional immunomodulators, which are agents that disturb or alter the immune system, so that an upward or downward regulation of humoral and / or mediated immunity is observed per cell. In one embodiment, upward regulation of the humoral and / or cell-mediated arms of the immune system is provided.
[0125] Examples of certain immunomodulators include, for example, an adjuvant or cytokine, or ISCOMATRIX ™ (CSL Limited; Parkville, Australia), described in U.S. Patent No. 5,254,339 among others. Non-limiting examples of adjuvants that can be used in the immunogenic composition of the present disclosure include the RIBI adjuvant system (Ribi Inc .; Hamilton, MT, USA), alum, mineral gels such as aluminum hydroxide gel, oil in water emulsions , water-in-oil emulsions, such as complete and incomplete Freund's adjuvants, block copolymer (CytRx; Atlanta, GA, USA), QS-21 (Cambridge Biotech Inc .; Cambridge, MA, USA), SAF-M (Chiron; Emeryville, CA, USA), adjuvant AMPHIGEN ™, saponin, Quil A or other fraction of saponin, lipid A monophosphoryl, and avridine (N, N-Dioctadecyl-N ', N'-bis (2-hydroxy ethyl) -1,3-diaminopropane, adjuvant of N-luteo-amine, N-Dioctadecyl-N ', N'-bis (2-hydroxy ethyl) propanediamine). Non-limiting examples of oil-in-water emulsions useful in the immunogenic composition of the disclosure include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN ™ 85 detergent (ICI Surfactants), 0.7% (v / 70 polysorbate detergent) v) (ICI Surfactants), 2.5% (v / v) ethanol, 200 mcg / ml of Quil A, 100 mcg / ml of cholesterol, and 0.5% (v / v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v / v) squalene, 1% (v / v) SPAN ™ 85 detergent, 0.7% (v / v) polysorbate detergent, 2.5% (v / v) ethanol, 100 mcg / ml Quil A, and 50 mcg / ml cholesterol. Other immunomodulators that can be included in the immunogenic composition include, for example, one or more interleukins, interferons, or other known cytokines or chemokines. In one embodiment, the adjuvant can be a cyclodextrin derivative or a polyanionic polymer, such as that described in Nos. 6,165,995 and U.S. 6,610,310, respectively. It should be understood that the immunomodulator and / or adjuvant to be used will depend on the individual to whom the immunogenic composition will be administered, the injection route and the number of injections to be applied.
[0126] The immunogenic compositions of the disclosure may further comprise one or more preservatives in addition to a plurality of capsular polysaccharide protein conjugates. The FDA requires that biological products in multi-dose (multi-dose) bottles contain a condom, with only a few exceptions. Vaccine products containing condoms include vaccines containing benzethonium chloride (anthrax), 2-phenoxy ethanol (DTaP, HepA, Lyme, Polio (parenteral)), phenol (Pneumo, Typhoid (parenteral), Smallpox) and thimerosal (DTaP, DT , Td, HepB, Hib, Flu, JE, Meningitis, Pneumonia, Rabies). Condoms approved for use in injectable drugs include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxy ethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal and phenyl mercuric nitrate. PACKAGING AND DOSAGE FORMS
[0127] The formulations of the disclosure may further comprise one or more between a buffer, a salt, a divalent cation, a nonionic detergent, a cryoprotectant, like a sugar, and an antioxidant, like a free radical scavenger or chelating agent, or any multiple combination thereof. The choice of any component, for example, a chelate, can determine whether another component (for example, a sequester) is desirable or not. The final composition formulated for administration must be sterile and / or pyrogen free. Individuals skilled in the art can empirically determine which combinations of these and other components will be optimal for inclusion in immunogenic compositions containing the disclosure preservative depending on a variety of factors such as the particular conditions of storage and administration required.
[0128] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more physiologically acceptable buffers selected, without limitation, from Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate . In certain embodiments, the formulation is buffered in a pH range from about 6.0 to about 9.0, preferably from about 6.4 to about 7.4.
[0129] In certain embodiments, it may be desirable to adjust the pH of the immunogenic composition or development formulation. The pH of a development formulation can be adjusted using standard techniques in the art. The formulation pH can be adjusted between 3.0 and 8.0. In certain embodiments, the pH of the formulation can be the same or can be adjusted to be between 3.0 and 6.0, 4.0 and 6.0, or 5.0 and 8.0. In other embodiments, the pH of the formulation can be the same or can be adjusted to be about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In certain modalities, the pH can be the same or can be adjusted to be in a range of 4.5 to 7.5, or from 4.5 to 6.5, from 5.0 to 5.4, from 5, 4 to 5.5, 5.5 to 5.6, 5.6 to 5.7, 5.7 to 5.8, 5.8 to 5.9, 5.9 to 6.0 , from 6.0 to 6.1, from 6.1 to 6.2, from 6.2 to 6.3, from 6.3 to 6.5, from 6.5 to 7.0, from 7.0 to 7.5 or 7.5 to 8.0. In a specific embodiment, the pH of the formulation is about 5.8.
[0130] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more divalent cations, including, without limitation, MgCh, CaCh and MnCh, in a concentration ranging from about 0.1 mM to about 10 mM, with up to about 5 mM being preferred.
[0131] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more salts, including, without limitation, sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, present in an ionic resistance that is physiologically acceptable to the individual through parenteral administration and included in a final concentration to produce a selected ionic resistance or osmolarity in the final formulation. The ionic resistance or final osmolality of the formulation will be determined by multiple components (for example, ions of buffering compound (s) and other non-buffering salts. A preferred salt, NaCI, is present in a range of up to about 250 mM , with saline concentrations being selected to complement other components (for example, sugars) so that the final total osmolarity of the formulation is compatible with parenteral administration (for example, intramuscular or subcutaneous injection) and will promote long-term stability of the immunogenic components of immunogenic composition formulation in various temperature ranges Salt-free formulations will tolerate increased ranges of one or more cryoprotectants selected to maintain desired final osmolarity levels.
[0132] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more cryoprotectants selected, without limitation, from disaccharides (eg lactose, maltose, sucrose or trehalose) and polyhydroxy hydrocarbons (eg , dulcitol, glycerol, mannitol and sorbitol).
[0133] In certain modalities, the osmolarity of the formulation is in a range of about 200 mOs / L to about 800 mOs / L, with a preferred range of about 250 mOs / L to about 500 mOs / L, or about 300 mOs / L to about 400 mOs / L. A salt-free formulation can contain, for example, from about 5% to about 25% sucrose, and preferably from about 7% to about 15%, or about 10% to about 12% sucrose. Alternatively, a salt-free formulation can contain, for example, from about 3% to about 12% sorbitol, and preferably from about 4% to 7%, or about 5% to about 6% sorbitol. If a salt such as sodium chloride is added, then the effective range of sucrose or sorbitol is relatively reduced. These and other considerations of osmolality and osmolarity are notorious in the art.
[0134] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more free radical oxidation inhibitors and / or chelating agents. A variety of free radical scavengers and chelates are known in the art and apply to the formulations and methods of use described in this document. Examples include, but are not limited to, ethanol, EDTA, an EDTA / ethanol combination, triethanolamine, mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, ascorbic acid / ascorbate, succinic acid / succinate, malic acid / maleate, desferal, EDDHA and DTPA, and various combinations of two or more among the above. In certain embodiments, at least one non-reducing free radical scavenger can be added in a concentration that effectively enhances the long-term stability of the formulation. One or more inhibitors of free radical oxidation / chelates can also be added in various combinations, such as a scavenger and a divalent cation. The choice of chelate will determine whether or not the addition of a sequester is necessary.
[0135] In certain embodiments, a disclosure formulation that is compatible with parenteral administration comprises one or more nonionic surfactants, including, without limitation, polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (TWEEN ™ 80), polysorbate- 60 (TWEEN ™ 60), polysorbate-40 (TWEEN ™ 40) and polysorbate-20 (TWEEN ™ 20), polyethylene ethyl esters, including, without limitation, Brij 58, Brij 35, as well as other TRITON ™ X-100 ; TRITON ™ X-114, NP40 (nonyl phenoxy polyethoxy ethanol), SPAN ™ 85 and the PLURONIC ™ series of non-ionic surfactants (eg PLURONIC ™ 121), with preferred polysorbate-80 components in a concentration of about 0.001 % to about 2% (up to about 0.25% being preferred) or polysorbate-40 in a concentration of about 0.001% to 1% (up to about 0.5% being preferred).
[0136] In certain embodiments, a disclosure formulation comprises one or more additional stabilizing agents suitable for parenteral administration, for example, a reducing agent comprising at least one thiol (-SH) group (for example, cysteine, N- acetyl cysteine, reduced glutathione, sodium thioglycolate, thiosulfate, monothioglycerol, or mixtures thereof). Alternatively or optionally, immunogenic composition formulations containing the preservative of the disclosure can be further stabilized by removing oxygen from the storage containers, protecting the formulation from light (for example, using amber glass containers).
[0137] Immunogenic composition formulations containing the preservative of the disclosure may comprise one or more pharmaceutically acceptable carriers or excipients, which include any excipient that does not induce an immune response. Suitable excipients include, but are not limited to, macromolecules, such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al, 2001, Vaccine, 19: 2118), trehalose, lactose and aggregates lipids (such as oil droplets or liposomes). These carriers are notorious for individuals skilled in the art. Pharmaceutically acceptable excipients are discussed, for example, in Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.
[0138] Developing compositions can be lyophilized or in aqueous form, that is, solutions or suspensions. Liquid formulations can advantageously be administered directly from their packaged form and, therefore, are ideal for injection without the need for reconstruction in aqueous medium as otherwise necessary for freeze-dried compositions of the development.
[0139] The direct distribution of immunogenic compositions of the present to an individual can be carried out by parenteral administration (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, or to the interstitial space of a tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. In a preferred embodiment, parenteral administration occurs by intramuscular injection, for example, in the individual's thigh or arm. The injection can take place through a needle (for example, a hypodermic needle), however, alternatively, a needle-free injection can be used. A typical intramuscular dose is 0.5 ml. Developing compositions can be prepared in various forms, for example, for injection as liquid solutions or suspensions. In certain embodiments, the composition can be prepared as a powder or spray for pulmonary administration, for example, in an inhaler. In other embodiments, the composition can be prepared as a suppository or pessary, or for nasal, aural or ocular administration, for example, as a spray, drops, gel or powder. Optimal amounts of components for a particular immunogenic composition can be confirmed by standard studies that involve the observation of appropriate immune responses in individuals. After an initial vaccination, individuals may receive one or more booster immunizations that are adequately spaced.
[0140] The immunogenic compositions of the disclosure may be packaged in a single dose or in a multiple dose form (eg, 2 doses, 4 doses, or more). For multiple dose forms, vials are typically, but not necessarily, preferred over pre-filled syringes. Suitable multiple dose formats include, but are not limited to: 2 to 10 doses per container at 0.1 to 2 ml per dose. In certain embodiments, the dose is a dose of 0.5 ml. See, for example, International Patent Application W02007 / 127668, which is incorporated herein by reference.
[0141] The compositions may be presented in vials or other suitable storage containers, or may be presented in pre-filled dispensing devices, for example, single component or multiple component syringes, which may or may not be equipped with needles. Typically, but not necessarily, a syringe contains a single dose of the immunogenic composition containing the disclosure preservative, although multiple pre-filled syringes are also considered. Similarly, a vial may include a single dose, but may alternatively include multiple doses.
[0142] Effective dosage volumes can be routinely established, but a typical dose of the composition for injection has a volume of 0.5 ml_. In certain embodiments, the dose is formulated for administration to a human subject. In certain modalities, the dose is formulated for administration to an adult human, young, adolescent, child or baby (that is, less than 1 year old) and can, in preferred modalities, be administered by injection.
[0143] Liquid immunogenic compositions of the disclosure are also suitable for reconstituting other immunogenic compositions that are present in lyophilized form. When an immunogenic composition needs to be used for this untimely reconstitution, the disclosure provides a kit with two or more vials, two or more ready-to-load syringes, or one or more of each, with the contents of the syringe being used to reconstitute the contents of the vial prior to injection, or vice versa.
[0144] Alternatively, the immunogenic compositions of the present disclosure can be lyophilized and reconstituted, for example, using one or more freeze-drying methods known in the art to form dry particles with regular shape (for example, spherical), such as micropellets or microspheres, having particle characteristics, such as medium diameter sizes that can be selected and controlled by varying the exact methods used to prepare them. The immunogenic compositions can further comprise an adjuvant which can optionally be prepared or contained in separate, regularly shaped (e.g. spherical) particles, such as micropellets or microspheres. In these embodiments, the present disclosure further provides an immunogenic composition kit comprising a first component that includes a stabilized dry immunogenic composition, optionally further comprising one or more preservatives from the disclosure, and a second component comprising a sterile aqueous solution. to reconstitute the first component. In certain embodiments, the aqueous solution comprises one or more preservatives, and may optionally comprise at least one adjuvant (see, for example, W02009 / 109550 (incorporated herein by reference)).
[0145] In yet another modality, a container of the multiple dose format is selected from one or more among the group consisting, without limitation, of generic laboratory glass bottles, beakers, graduated cylinders, fermenters, bioreactors, pipes, tubes, bags, jars, conceptacles, caps for conceptacles (for example, a rubber stopper, a screw on the cap), ampoules, syringes, double or multiple chamber syringes, syringe plugs, syringe plungers, rubber caps, caps plastic, glass lids, disposable cartridges and pens, and the like. The container of the present disclosure is not limited to the material of manufacture, and includes materials such as glass, metals (for example, steel, stainless steel, aluminum, etc.) and polymers (for example, thermoplastics, elastomers, thermoplastic elastomers). In a particular embodiment, the shape container is a 5 ml Schott Type 1 glass conceptacle with a butyl stopper. The skilled artisans will appreciate that the format presented above is in no way an exhaustive list, but merely serves as a guideline to the artisan regarding the variety of formats available for the present revelation. Additional formats contemplated for use in the present disclosure can be found in published catalogs from vendors and manufacturers of laboratory equipment, such as United States Plastic Corp. (Lima, OH, USA), VWR. METHODS FOR INDUCING AN IMMUNE RESPONSE AND PROTECTION AGAINST INFECTION
[0146] The present disclosure also includes methods of use for immunogenic compositions described in this document. For example, one embodiment of the disclosure provides a method for inducing an immune response against a pathogenic bacterium, for example, S. pneumonia, which comprises administering to an individual an immunogenic amount of any of the immunogenic compositions described herein that comprise an antigen. bacterial, like a bacterial capsular polysaccharide derived from pathogenic bacteria. One embodiment of the disclosure provides a method to protect an individual against infection by S. pneumoniae, or a method to prevent infection by S. pneumoniae, or a method to reduce the severity or delay of the principle of at least one symptom associated with an infection. caused by S. pneumoniae, the methods comprising administering to an individual an immunogenic amount of any of the immunogenic compositions described herein that comprise a bacterial antigen, such as a bacterial capsular polysaccharide derived from S. pneumoniae. disclosure provides a method for treating or preventing a Streptococcal infection, disease or condition associated with a Streptococcus sp. in an individual, the method comprising the step of administering a therapeutically or prophylactically effective amount of an immunogenic composition described herein to the individual. In some embodiments, the method for treating or preventing a Streptococcal infection, disease or condition comprises human, veterinary, animal, or agricultural treatment. Another modality provides a method for treating or preventing a Streptococcal infection, disease or condition associated with a Streptococcus sp. in an individual, the method comprising generating a polyclonal or monoclonal antibody preparation from the immunogenic composition described herein, and using said antibody preparation to impart passive immunity to the individual. One embodiment of the disclosure provides a method for preventing a Streptococcal infection in an individual undergoing a surgical procedure, the method comprising the step of administering a prophylactically effective amount of an immunogenic composition described in the present document to the individual prior to the surgical procedure.
[0147] An "immune response" to an antigen or immunogenic composition is the development in an individual of a humoral and / or cell-mediated immune response to molecules present in the antigen or vaccine composition of interest. For the purposes of the present disclosure, a “humoral immune response” is an antibody-mediated immune response and involves the induction and generation of antibodies that recognize and agglutinate with some affinity for the antigen in the immunogenic composition of the disclosure, while a “mediated immune response” per cell ”is that mediated by T cells and / or other white blood cells. A “cell-mediated immune response” is caused by the presentation of antigenic epitopes in association with Class I or Class II molecules of the major histocompatibility complete (MHC), CD1 or other non-classical MHC type molecules. This activates antigen-specific helper CD4 + T cells or CD8 + cytotoxic Y lymphocyte cells (“CTLs”). CTLs have specificity for peptide antigens that are presented in association with problems encoded by classical or non-classical MHCs and expressed on cell surfaces. CTLs help to induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with these microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T cells. Helper T cells act to help stimulate the function, and focus, of nonspecific effector cells against cells that exhibit peptide or other antigens in association with classical or non-classical MHC molecules on their surface. A "cell-mediated immune response" also refers to the production of cytokines, chemokines and other molecules produced by activated T cells and / or other white blood cells, including those derived from CD4 + CD8 + T cells. The ability of a particular antigen or composition to stimulate a cell-mediated immune response can be determined by a series of assays, such as lymphoproliferation assays (lymphocyte activation), CTL cytotoxic cell assays, by the assay for specific T lymphocytes for the antigen in a sensitized individual, or by measuring cytokine production by T cells in response to antigen re-stimulation. These tests are notorious in the art. See, for example, Erickson et al. (1993) J. Immunol. 151: 4189-4199; and Doe et al. (1994) Eur. J. Immunol.24: 2369-2376.
[0148] Depending on the use in question, "treatment" (including variations thereof, for example, "treat" or "treated (a)") means any one or more of the following: (i) prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction in severity, or, the elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen or disorder in question. Therefore, treatment can be carried out prophylactically (before infection) or therapeutically (after infection). In the present disclosure, prophylactic treatment is the preferred mode. In accordance with a particular embodiment of the present disclosure, compositions and methods are provided that treat, including prophylactically and / or therapeutically immunizing a host animal against a microbial infection (for example, a bacterium such as Streptococcus). The methods of the present disclosure are useful to confer prophylactic and / or therapeutic immunity to an individual. The methods of the present disclosure can also be practiced on individuals for biomedical research applications.
[0149] Depending on the use in question, "mammal" means a human or non-human animal. More particularly, a mammal refers to any animal classified as a mammal, including humans, domestic and farm animals, and research, zoo, sports and domestic animals, such as a pet and other domesticated animals including, without limitation, cattle, sheep, ferrets, pigs, horses, rabbits, goats, dogs, cats, and the like. The preferred pets are dogs and cats. Preferably, the mammal is a human being.
[0150] An “immunogenic amount,” and an “immunologically effective amount,” both being used interchangeably in this document, refer to an amount of antigen or immunogenic composition sufficient to elicit an immune response, be it a cellular response (cell T) or humoral (B cell or antibody), or both, as measured by standard assays known to a person skilled in the art.
[0151] The amount of a particular conjugate in a composition is generally calculated based on the total, conjugated and unconjugated polysaccharide for that conjugate. For example, a conjugate with 20% free polysaccharide will have about 80 mcg of conjugated polysaccharide and about 20 mcg of unconjugated polysaccharide in a dose of 100 mcg polysaccharide. The protein contribution to the conjugate is generally not considered when calculating the dose of a conjugate. The amount of conjugate may vary depending on the streptococcal serotype. In general, each dose will comprise 0.1 to 100 mcg of polysaccharide, particularly 0.1 to 10 mcg, and, more particularly, 1 to 10 mcg. The "immunogenic amount" of the different polysaccharide components in the immunogenic composition may differ and each may comprise 1 mcg, 2 mcg, 3 mcg, 4 mcg, 5 mcg, 6 mcg, 7 mcg, 8 mcg, 9 mcg, 10 mcg , 15 mcg, 20 mcg, 30 mcg, 40 mcg, 50 mcg, 60 mcg, 70 mcg, 80 mcg, 90 mcg, or about 100 mcg of any particular polysaccharide antigen.
[0152] An "invasive disease" of S. pneumoniae is the isolation of bacteria from a normally sterile site, where there are associated clinical signs / symptoms of the disease. Normally sterile body sites include blood, CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint / synovial fluid, bone, internal body site (lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, ovary ) or other normally sterile sites. Clinical conditions that characterize invasive diseases include bacteremia, pneumonia, cellulite, osteomyelitis, endocarditis, septic shock, and the like.
[0153] The effectiveness of an antigen as an immunogen can be measured either by proliferation assays, cytolytic assays, such as chromium release assays to measure the ability of a T cell to lyse its specific target cell, or by measuring the levels of B cell activity by measuring the levels of circulating antibodies specific for the antigen in the serum. An immune response can also be detected by measuring serum levels of antigen-specific antibody induced after administration of the antigen, and, more specifically, by measuring the ability of the antibodies thus induced to enhance the opsonophagocytic capacity of particular white blood cells. , as described in this document. The level of protection of the immune response can be measured by challenging the host immunized with the antigen that was administered. For example, if the antigen to which an immune response is desired is a bacterium, the level of protection induced by the immunogenic amount of the antigen is measured by detecting percent survival or percent mortality after challenge of animals with bacterial cells. In one embodiment, an amount of protection can be measured by measuring at least one symptom associated with bacterial infection, for example, a fever associated with infection. The amount of each of the antigens in the multi-antigen or multi-component vaccine or immunogenic compositions will vary with respect to each of the other components and can be determined by methods known to the skilled person. Such methods would include procedures for measuring immunogenicity and / or effectiveness in vivo. In certain embodiments, the term “about” means within 20%, preferably within 10%, and more preferably within 5% of the indicated value or range.
[0154] The disclosure further provides antibodies and antibody compositions that agglutinate specifically and selectively to the capsular or glycoconjugate polysaccharides of the present disclosure. In some embodiments, antibodies are generated by administering the capsular polysaccharides or glycoconjugates of this disclosure to an individual. In some embodiments, the disclosure provides purified or isolated antibodies directed against one or more of the capsular or glycoconjugate polysaccharides of the present disclosure. In some embodiments, the antibodies of the present disclosure are functional as measured by killing bacteria in an animal efficacy model or through an opsonophagocytic extermination assay. In some embodiments, the antibodies in the disclosure confer passive immunity to an individual. The present disclosure further provides polynucleotide molecules that encode an antibody or antibody fragment of the disclosure, and a cell, cell line (such as hybridoma cells or other engineering modified cell lines for recombinant antibody production) or a transgenic animal that produce an antibody or developing antibody composition, using techniques notorious for individuals skilled in the art.
[0155] The antibodies or antibody compositions of the disclosure can be used in a method to treat or prevent a staphylococcal infection, disease or condition associated with a Streptococcus sp. in an individual, the method comprising generating a polyclonal or monoclonal antibody preparation, and using said antibody or antibody composition to impart passive immunity to the individual. The antibodies in the disclosure may also be useful for diagnostic methods, for example, detecting the presence or quantifying the levels of capsular polysaccharide or a glycoconjugate thereof.
[0156] The following examples are offered by way of illustration and without limitation. Abbreviations: PM = molecular weight; WFI = water for injection; TEMPO = 2,2,6,6-Tetramethyl-1-piperidinyloxy free radical; NCS = N-Chlorosuccinimide. EXAMPLES
Example 1: Conjugation of Pn serotype-12F using TEMPO / NCS [0157] In order to improve the stability of serotype 12F-CRM-I97 glycoconjugates, alternative chemicals were explored using 2,2,6,6-Tetramethyl free radical -1-piperidinyloxy (TEMPO) and N-Chlorosuccinimide (NCS) as the co-oxidant to oxidize primary alcohols in aldehyde groups. GC / MS analysis showed that the oxidation sites were different from those of periodate-mediated oxidation. In the case of TEMPO-NCS oxidation, a-D-GIcp and 2-Glcp were oxidized, while a-D-Galp was the main oxidation site when the periodate was used (see Figure 1). As described in more detail, TEMPO was used in catalytic amounts (<0.1 molar equivalent) and the desired degree of oxidation (OD) was achieved by varying the amounts of NCS used. Subsequently, several conjugates were synthesized and characterized. In general, the production of serotype 12F glycoconjugates was carried out in several stages, as follows:
[0158] 1) Hydrolysis of serotype 12 polysaccharide in molecular weights 50 to 500 kDa.
[0159] 2) Activation of serotype 12F polysaccharide with TIME / NCS
[0160] 3) Purification of activated polysaccharide
[0161] 4) 12F serotype conjugation to CRM197 protein
[0162] 5) Purification of 12F-CRM serotype conjugates. Example 2: Hydrolysis and oxidation of serotype 12F
[0163] Polysaccharide hydrolysis was typically performed under acidic conditions with heating to obtain an average molecular weight in the desired range of 100 to 350 kDa. A typical experiment is described below. Hydrolysis
[0164] The serotype 12F polysaccharide solution was added to a jacketed reaction vessel. To this, the required volume of 0.30 M acetic acid and water for injection (WFI) was added to maintain a concentration of acetic acid at - 0.1 M. The pH of the solution was adjusted to 3.2 ± 0, 3 used 1 N NaOH or glacial acetic acid. The temperature of the reaction mixture was raised to 70 ± 5 ° C. The reaction mixture was stirred at 70 ± 5 ° C for 90 to 120 minutes. The reaction mixture was cooled to 23 ± 2 ° C and neutralized (pH 7.0) by adding a 1 M NaOH solution. The hydrolyzed polysaccharide was purified by ultrafiltration / diafiltration against WFI using 30K MWCO membranes. The solution was filtered through a 0.22 pm filter and stored at 2 to 8 ° C until oxidation. The molecular weight of the hydrolyzed polysaccharide was analyzed by SEC-MALLS to ensure that the molecular weight satisfies the target range of 100 to 350 kDa. Partial oxidation
[0165] In one experiment, the serotype 12F polysaccharide was mechanically sized using pressure homogenization using a microfluidizer to reduce the molecular weight to approximately 100 to 500 kDa. The dimensioned polysaccharide was added to a reaction vessel at a concentration of 4.0 mg / mL and mixed with bicarbonate / carbonate buffer (0.5 M NaHCOa / 0.05 M Na2CO3 buffer, pH 8.6) in a 1: 1 v / v ratio. To the stirred mixture, <0.1 molar equivalent of TEMPO was added. The reaction was initiated by the addition of 0.6 to 1.0 molar equivalent of NCS. The reaction mixture was stirred at room temperature for 2 hours, after which the activated polysaccharide was purified by diafiltration, with WFI using a 30K ultrafiltration membrane. The purified polysaccharide was collected and the degree of oxidation (OD) determined by quantitative measurements of aldehyde (using a 3-methyl-2-benothiazolinone hydrazone (MBTH) assay) and polysaccharide (using an anthrone assay).
[0166] In another experiment, the serotype 12F polysaccharide was hydrolyzed to reduce the molecular weight to a molecular weight of approximately 100 to 500 kDa. The serotype 12F polysaccharide was added to a reaction vessel and mixed with a 0.5 M NaHCOa / 0.05 M Na2COa (pH 8.6) buffer in a 1: 1 v / v ratio. To the stirred mixture was added 0.6 to 1.0 molar equivalent of NCS dissolved in WFI. Activation was initiated by adding approximately 0.1 molar equivalent of TEMPO dissolved in WFI. The reaction mixture was stirred at room temperature for 2 hours, after which the activated polysaccharide was purified by diafiltration with WFI using a 30K ultrafiltration membrane. The purified activated polysaccharide was filtered through a 0.2 pm filter and stored at 4 ° C before use.
[0167] TEMPO / NCS-mediated oxidations have also been carried out successfully in sodium phosphate buffers of pH 6.5, 7.0, 7.5 and 8.0. In some activation experiments, a primary alcohol, such as n-propanol, was used to cool the reagents in order to prevent saccharide over-oxidation. In another set of experiments, the chemically hydrolyzed polysaccharide was subjected to oxidation directly, without the purification step by ultrafiltration / diafiltration. Example 3: Conjugation of 12F serotype oxidized polysaccharide
[0168] In one experiment, the purified oxidized 12F serotype polysaccharide was added to a reaction vessel followed by the addition of a 0.5 M sodium phosphate buffer (pH 6.5) at a final buffer concentration of 0 , 1 M. To this solution, CRM197 previously lyophilized was added and mixed vigorously in order to obtain a homogeneous solution. The pH was adjusted to 6.8 using a diluted solution of HCl or 1N NaOH. This was followed by the addition of 1.5 molar equivalent of NaCNBHa. The reaction mixture was stirred for 24 hours at room temperature (23 ° C) and for 2.5 days at 37 ° C. The reaction mixture was then diluted with a 1X 0.9% saline solution and the unreacted aldehyde groups were "capped" with 2 molar equivalents of sodium borohydride. The capping reaction time was 3 hours.
[0169] In another experiment, the purified activated Serotype 12F was added to a reaction vessel followed by the addition of 0.5 M sodium phosphate buffer (pH 6.5) to a final buffer concentration of 0.1 M To this solution, CRM197 previously lyophilized was added and mixed vigorously to obtain a homogeneous solution. The pH was adjusted to 6.8 using a diluted solution of HCl or 1N NaOH. This was followed by the addition of 3 molar equivalents of NaCNBHa. The reaction mixture was stirred for 24 hours at 23 ° C and for 48 hours at 37 ° C. The reaction mixture was then diluted with a 1X 0.9% saline solution and, with stirring, the unreacted aldehyde groups were "capped" with 1 molar equivalent of sodium borohydride NaBH4. The capping reaction time was 3 hours.
[0170] In another experiment, the purified activated 12F serotype was added to a reaction vessel and mixed with a CRM197 solution. The mixture was lyophilized and the powder dissolved in a 0.1 M sodium phosphate buffer (pH 6.8) at a final saccharide concentration of 5 mg / ml. If necessary, the pH was adjusted to 6.8 using a diluted solution of HCl or 1N NaOH. This was followed by the addition of 3 molar equivalents of NaCNBHs. The reaction mixture was stirred for 24 hours at 23 ° C and for 48 hours at 37 ° C. The reaction mixture was then diluted with a 1X 0.9% saline solution, the unreacted aldehyde groups were “capped” with 1 molar equivalent of sodium borohydride NaBH4. The capping reaction time was 3 hours. Example 4: Purification of conjugate
[0171] The capped reaction mixture was filtered using a 5 pm filter and then purified using MWCO 100K ultrafiltration membranes. The conjugate was first diafiltered using a 10 mM succinate / 0.9% saline buffer, pH 6.0. The purified conjugate was then filtered through 0.45 / 0.22 pm filters to obtain a bulk conjugate. Example 5: Degree of oxidation
[0172] A successful oxidation of primary alcohols in the serotype 12F polysaccharide was achieved using the TEMPO / NCS system. The hydrolyzed serotype 12F polysaccharides were oxidized to varying degrees of oxidation levels (OD) by adjusting an amount of NCS co-oxidant. The effect on OD by varying the amounts of NCS using different polysaccharide lots and molecular weights is shown in Figure 2. Typically, 0.5 to 2.5 molar equivalents of NCS were used to achieve the desired degree of oxidation. Typically, the oxidation reaction is complete in 2 hours as no significant change in OD was observed after 2 hours.
[0173] Several serotype 12F conjugates have been generated and characterized using the TEMPO / NCS oxidized polysaccharide. The results are summarized in Table 1. Some representative conjugates have also been successfully generated using other active pneumococcal serotypes with TEMPO / NCS system. The procedure for generating conjugates for other pneumococcal serotypes was the same as the method used for Serotype 12F. The results are described in Tables 2 to 4.
Exemplo 7: Mecanismo putativo para o conjugado de Pn-serotipo 12F usando radical nitroxila na presença de um oxidante, como TEMPO/NCS[0174] The titers of opsonophagocytic activity (OPA) for serotype 12F-CRM197 conjugates in mice were determined in mice under standard conditions. OPA titles (geometric mean title (GMT) with a 95% confidence interval (CI) at four and seven weeks are shown in Table 5, demonstrating that the serotype I2F-CRM197 conjugate (Lot 12F-97B; see also Table 1 for characterization data for this conjugate) caused OPA titers in a murine immunogenicity model. The conjugate generated by TEMPO-NCS was more immunogenic than the control conjugate (171B) generated from the oxidation of periodate.Table 5: Immunogenicity of serotype 12F-CRM197 conjugates Example 7: Putative mechanism for the Pn-serotype 12F conjugate using nitroxyl radical in the presence of an oxidant, such as TEMPO / NCS
[0175] The putative Pn-serotype 12F oxidation / conjugation mechanism is shown in Figure 6. The primary hydroxyl groups of the polysaccharide are oxidized by catalytic amounts of nitroxyl radical, such as TEMPO, with an oxidizer, such as NCS as the stoichiometric oxidant . The current oxidant is the N-oxoammonium salt, in a catalytic cycle. The oxidation of the primary hydroxyl groups C-6 generates aldehyde groups that are subsequently reacted with the primary amino groups of the carrier protein lysine (CRM197) to generate the glycoconjugate. Example 8: Comparison of stability
[0176] Comparing the stability of (at 25 ° C) conjugates generated by oxidation of periodate vs oxidation of TEMPO / NCS (see Figure 7) demonstrated that the conjugate generated by oxidation of the Pn-12F polysaccharides was relatively more stable. As shown in Figure 7, an increase in free saccharide over time has been observed for the glycoconjugate generated by the oxidation of the Pn-12F polysaccharide periodate at 25 ° C. In contrast, the glycoconjugate prepared using the TEMPO / NCS oxidation of the Pn-12F polysaccharide did not show significant trends for free saccharide under similar conditions.

权利要求:
Claims (19)
[0001]
1. Method for producing a glycoconjugate that comprises a saccharide conjugated to a carrier protein, characterized by the fact that it comprises the steps of: reacting a saccharide, in which said saccharide is a bacterial capsular polysaccharide, with 2,2,6,6 -tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and reacting the activated saccharide with a carrier protein comprising one or more amino groups.
[0002]
2. Method according to claim 1, characterized in that the saccharide is reacted with 0.1 to 10 molar equivalent of N-chlorosuccinimide.
[0003]
3. Method according to claim 1, characterized by the fact that the saccharide is reacted with 0.5 to 1.5 molar equivalent of N-chlorosuccinimide.
[0004]
Method according to any one of claims 1 to 3, characterized by the fact that 2,2,6,6-tetramethyl-1-piperidineyloxy (TEMPO) is present in a catalytic amount.
[0005]
Method according to any one of claims 1 to 3, characterized in that the saccharide is reacted with less than 0.3 molar equivalent of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).
[0006]
Method according to any one of claims 1 to 5, characterized in that the degree of oxidation of the activated saccharide varies from 3 to 40.
[0007]
Method according to any one of claims 1 to 5, characterized in that the degree of oxidation of the activated saccharide varies from 6 to 14.
[0008]
Method according to any one of claims 1 to 7, characterized in that the capsular polysaccharide is derived from S. pneumoniae.
[0009]
9. Method according to claim 8, characterized by the fact that the capsular polysaccharide is selected from capsular polysaccharides of Pn-serotype 3, Pn-serotype 10A, Pn-serotype 12F, and Pn-serotype 33F.
[0010]
Method according to any one of claims 1 to 7, characterized in that the capsular polysaccharide is derived from N. meningitidis.
[0011]
11. Method according to claim 10, characterized by the fact that the capsular polysaccharide is selected from meningococcal capsular polysaccharides (Mn) -serotype A, C, W135, and capsular polysaccharides Y.
[0012]
12. Method according to claim 10, characterized by the fact that the capsular polysaccharide is capsular polysaccharide (Mn) -serotype X.
[0013]
13. Method according to any one of claims 1 to 12, characterized in that the carrier protein is a toxin from tetanus, diphtheria, whooping cough, Pseudomonas, E. coli, Staphylococcus or Streptococcus.
[0014]
14. Method according to any one of claims 1 to 12, characterized in that the carrier protein is CRM197.
[0015]
15. Method according to any one of claims 1 to 14, characterized by the fact that before step a) the saccharide is dimensioned.
[0016]
16. Method according to any one of claims 1 to 14, characterized by the fact that before step a) the saccharide is hydrolyzed or mechanically dimensioned.
[0017]
17. Method according to any one of claims 1 to 14, characterized by the fact that before step a) the saccharide is hydrolyzed or mechanically sized by pressure homogenization to reach a molecular weight of 50 kDa to 500 kDa.
[0018]
18. Method according to any one of claims 1 to 14, characterized in that before step a) the saccharide is hydrolyzed to a molecular weight ranging from 100 to 400 KDa.
[0019]
19. Method according to any one of claims 1 to 14, characterized by the fact that before step a) saccharide is hydrolyzed, at a molecular weight ranging from 150 to 350 KDa.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07H 3/06 (2006.01), A61K 47/00 (2006.01), C07K 1/ |

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-10-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-05-26| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-07-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/12/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261740311P| true| 2012-12-20|2012-12-20|

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US61/740,311|2012-12-20|

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PCT/IB2013/060933|WO2014097099A2|2012-12-20|2013-12-13|Glycoconjugation process|

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