a method of making a glycoconjugate comprising a saccharide conjugated to a carrier protein by means

专利摘要:
GLYCOCONJUGATION PROCESSES AND COMPOSITIONS. The invention provides eTEC-linked glycoconjugates that comprise a saccharide covalently conjugated to a carrier protein by means of a (2 - ((2-oxoethyl) thio) ethyl) carbamate (ETEC) spacer, immunogenic compositions comprising such glycoconjugates and methods for the preparation and use of these glycoconjugates and immunogenic compositions.
公开号:BR112015003227B1
申请号:R112015003227-3
申请日:2013-08-12
公开日:2020-10-27
发明作者:Jianxin Gu；Jin-Hwan Kim；A.Krishna Prasad；Yu-Ying Yang
申请人:Pfizer Inc.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[0001] The invention generally relates to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein by means of a spacer (2 - ((2-oxoethyl) thio) ethyl) carbamate (eTEC), to compositions immunogenic substances comprising such glycoconjugates and methods for preparing and using such glycoconjugates and immunogenic compositions. BACKGROUND OF THE INVENTION
[0002] The approach to increase the immunogenicity of weakly immunogenic molecules by conjugating these molecules to "carrier" molecules has been used successfully for decades (see, for example, Goebel et al. (1939) J. Exp. Med. 69: 53). For example, many immunogenic compositions have been described in which purified capsular polymers have been conjugated to carrier proteins to create more effective immunogenic compositions in exploiting this "carrier effect" (Schneerson et al. (1984) Infect. Immun. 45: 582-591). It has also been shown that overcomes the weak antibody response normally seen in infants when immunized with a free polysaccharide (Anderson et al. (1985) J. Pediatr. 107: 346; Insel et al. (1986) J. Exp. Med. 158: 294).
[0003] Conjugates have been successfully generated using various crosslinking or coupling reagents, such as homobifunctional, heterobifunctional or zero length crosslinking agents. Several methods are available for coupling immunogenic molecules, such as saccharides, proteins and peptides, to peptide or protein carriers. Most methods create amine, amide, urethane, isothiourea or disulfide bonds or, in some cases, thioethers. A disadvantage in using crosslinking or coupling reagents which introduce reactive sites into the side chains of reactive amino acid molecules on the carrier and / or immunogenic molecules is that the reactive sites, if not neutralized, are free to react with any unwanted molecules, either in vitro (thus, potentially adversely affecting the functionality or stability of the conjugates) or in vivo (thus, representing a potential risk of adverse events in people or animals immunized with the preparations). Such excess reactive sites can be reacted or "capped" in order to inactivate these sites using various known chemical reactions, but these reactions can otherwise be detrimental to the functionality of the conjugates. This can be particularly problematic when trying to create a conjugate by introducing reactive sites in the carrier molecule, since its larger size and more complex structure (in relation to the immunogenic molecule) can make it more vulnerable to the negative effects of chemical treatment. . Thus, there remains a need for new methods to prepare carrier protein conjugates with an appropriate "cap", so that vehicle functionality is preserved and the conjugate retains the ability to elicit the desired immune response. SUMMARY OF THE INVENTION
[0004] The present invention is directed to methods of preparing glycoconjugates that comprise a saccharide covalently conjugated to a carrier protein via a bivalent hetero-functional ligand referred to herein as an ethyl (2 - (((2-oxoethyl) thio) spacer) carbamate (ETEC). The eTEC spacer includes seven linear atoms (ie, -C (O) NH (CH2) 2SCH2C (O) -) and provides stable thioether and amide bonds between the carrier protein and the saccharide. The invention further provides eTEC-linked glycoconjugates, immunogenic compositions comprising them and methods for using such glycoconjugates and immunogenic compositions.
[0005] In one aspect, the invention provides a glycoconjugate comprising a saccharide conjugated to a carrier protein by means of an eTEC spacer, wherein the saccharide is covalently attached to the eTEC spacer via a carbamate bond and in which the carrier protein is covalently linked to the eTEC spacer via an amide bond.
[0006] In some embodiments, saccharide is a polysaccharide, such as a capsular polysaccharide derived from bacteria, in particular from pathogenic bacteria. In other embodiments, the saccharide is an oligosaccharide or a monosaccharide. The eTEC-linked glycoconjugates of the invention can be represented by the general formula (I):

[0007] in which the atoms comprising the eTEC spacer are contained (I),
[0008] in which the atoms comprising the eTEC spacer are contained in the central rectangle.
[0009] The carrier proteins incorporated in the glycoconjugates of the invention are selected from the group of carrier proteins generally suitable for such purposes, as further described herein or known to those skilled in the art. In particular modalities, the carrier protein is CRM197.
[00010] In another aspect, the invention provides a method of manufacturing a glycoconjugate comprising a saccharide conjugated to a carrier protein by means of an eTEC spacer comprising the steps of: a) reacting a saccharide with a carbonic acid derivative in an organic solvent to produce an activated saccharide; b) reaction of the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first "capping" reagent capable of "capping" unconjugated α-haloacetamide groups from the activated carrier protein; and / or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide; wherein an eTEC-linked glycoconjugate is produced.
[00011] In frequent embodiments, the carbonic acid derivative is 1,1'-carbonyl-di- (1,2,4-triazole) (CDT) or 1, T-carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). In preferred embodiments, the thiolated saccharide is produced by reacting the activated saccharide with the symmetrical bifunctional reagent thioalkylamine, cystamine or a salt thereof. Alternatively, the thiolated saccharide can be formed by reacting the activated saccharide with cysteamine or a salt thereof. The glycoconjugates bound by eTEC produced by the invention methods can be represented by the general formula (I).
[00012] In frequent modalities, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamide groups from the lysine residues of the carrier protein to form an S-carboxymethylcysteine (CMC) residue ) covalently attached to the activated lysine residue via a thioether bond. In other embodiments, the second "capping" reagent is iodoacetamide (IAA), which reacts with free unconjugated sulfhydryl groups of the activated thiolated saccharide to provide a "cap-ped" thioacetamide. Often, step e) comprises both capping with a first capping reagent and a second capping reagent. In certain embodiments, step e) comprises "capping" with N-acetyl-L-cysteine as the first reagent and "capping" with IAA as the second capping reagent.
[00013] In some modalities, the capping step e) further comprises reaction with a reducing agent, for example, DTT, TCEP or mercaptoethanol, after reaction with the first and / or second capping reagent.
[00014] In some embodiments, step d) further comprises providing an activated carrier protein comprising one or more α-haloacetamide groups prior to reaction of the activated thiolated saccharide with the activated carrier protein. In frequent embodiments, the activated carrier protein comprises one or more a-bromoacetamide groups.
[00015] In another aspect, the invention provides a glycoconjugate comprising an eTEC-linked saccharide conjugated to a carrier protein by means of an eTEC spacer produced according to any of the methods described herein.
[00016] For each aspect of the invention, in particular embodiments of the methods and compositions described here, the glycoconjugate bound by eTEC comprises a saccharide which is a bacterial capsular polysaccharide, in particular a capsular polysaccharide derived from pathogenic bacteria .
[00017] In some of these modalities, the eTEC-linked glycoconjugate comprises a pneumococcal capsular polysaccharide (Pn) derived from Streptococcus pneumoniae. In specific modalities, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.
[00018] In other embodiments, the eTEC-linked glycoconjugate comprises a meningococcal capsular polysaccharide (Mn) derived from Neisseria meningitidis. In specific embodiments, the capsular polysaccharide Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y.
[00019] In particularly preferred embodiments, saccharide is a bacterial capsular polysaccharide, such as a Pn or Mn capsular polysaccharide, covalently conjugated to CRM197 by means of an eTEC spacer.
[00020] The compositions and methods described here are useful in a variety of applications. For example, the glycoconjugates of the invention can be used in the production of immunogenic compositions comprising an eTEC-linked glycoconjugate. Such immunogenic compositions can be used to protect receptors against bacterial infections, for example, by pathogenic bacteria, such as S. pneumoniae or N. meningitidis.
[00021] Thus, in another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a saccharide covalently conjugated to a carrier protein by means of an eTEC spacer , as described here.
[00022] In frequent embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide.
[00023] In some of these embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate which comprises a S. pneumoniae-derived capsular polysaccharide. In some specific embodiments, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.
[00024] In other embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising an Mn capsular polysaccharide derived from N. meningitidis. In some specific embodiments, the capsular polysaccharide Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135eY.
[00025] In preferred embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide, such as a Pn or Mn capsular polysaccharide, covalently conjugated to CRM197 by means of an eTEC spacer.
[00026] In some embodiments, the immunogenic compositions comprise an adjuvant. In some of these 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 compositions described herein comprise the aluminum phosphate adjuvant.
[00027] In another aspect, the invention provides a method of preventing, treating or ameliorating an infection, disease or bacterial condition in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition of the invention, wherein the said immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial antigen, such as a bacterial capsular polysaccharide.
[00028] In one embodiment, the infection, disease or condition is associated with S. pneumoniae bacteria and the glycoconjugate comprises a capsular polysaccharide Pn. In another embodiment, the infection, disease or condition is associated with bacteria N. meningitidis and the glycoconjugate comprises a capsular polysaccharide Mn.
[00029] In other respects, the invention provides a method for inducing an immune response against pathogenic bacteria; a method for preventing, treating or ameliorating a disease or condition caused by pathogenic bacteria; and a method for reducing the severity of at least one symptom of an infection, disease or condition caused by pathogenic bacteria, in each case by administering to an individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate. and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial antigen, such as a bacterial capsular polysaccharide derived from pathogenic bacteria.
[00030] In another aspect, the invention provides a method of inducing an immune response in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically-excipient, carrier or diluent acceptable, wherein the glycoconjugate comprises a bacterial antigen, such as a bacterial capsular polysaccharide. In preferred fashion, the method involves producing a protective immune response in the individual, as further described here.
[00031] In another aspect, the invention provides a method of administering an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate to an individual to generate a protective immune response in the individual, as further described herein.
[00032] In another aspect, the invention provides an antibody generated in response to an eTEC-linked glycoconjugate of the present invention or an immunogenic composition comprising such glycoconjugate. Such antibodies can be used in research and clinical laboratory tests, such as detection and serotyping of bacteria, or they can be used to confer passive immunity on an individual.
[00033] In yet another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate of the present invention for use in preventing, treating or ameliorating bacterial infection, for example, infection by S. pneumoniae or N. meningitidis.
[00034] In another aspect, the invention provides the use of an immunogenic composition comprising an eTEC-linked glycoconjugate of the present invention for the preparation of a medicament for the prevention, treatment or amelioration of bacterial infection, for example, infection by S. pneumoniae or N. meningitidis.
[00035] In certain preferred embodiments of the therapeutic and / or prophylactic methods and uses described above, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide covalently linked to a carrier protein by means of an eTEC spacer. In frequent modalities of the methods and uses described here, the bacterial capsular polysaccharide is a Pn capsular polysaccharide or an Mn capsular polysaccharide. In some of these modalities, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 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 Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y.
[00036] In certain preferred embodiments, the carrier protein is CRM197. In particularly preferred embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide, such as a Pn or Mn capsular polysaccharide, covalently conjugated to the CRM 197 by means of an eTEC spacer. BRIEF DESCRIPTION OF THE DRAWINGS
[00037] Figure 1 shows a general scheme for the preparation of the eTEC-linked glycoconjugates of the invention for a glycoconjugate comprising a polysaccharide covalently conjugated to CRM197.
[00038] Figure 2 shows a polysaccharide repeat structure of S. pneumoniae serotype 33F (33F-Pn) capsular polysaccharide.
[00039] Figure 3 shows a polysaccharide repeat structure of S. pneumoniae serotype 22F (22F-Pn) capsular polysaccharide.
[00040] Figure 4 shows a polysaccharide repeat structure of S. pneumoniae serotype 10A (10A-Pn) capsular polysaccharide.
[00041] Figure 5 shows a polysaccharide repeat structure of S. pneumoniae serotype 11A (11A-Pn) capsular polysaccharide.
[00042] Figure 6 shows a representative structure of a Pn-33F glycoconjugate that incorporates the eTEC ligand (A) and potential free sulfhydryl sites (B) with "cap" and without "cap".
[00043] Figure 7 shows a flowchart of the representative process for the activation (A) and conjugation (B) processes used in the preparation of the glycoconjugate Pn-33F to CRM197.
[00044] Figure 8 shows a thiol level of capsular polysaccharide Pn-33F as a function of molar equivalents of CDT used in the activation step. DETAILED DESCRIPTION
[00045] The present invention can be more easily understood by reference to the following detailed description of the preferred embodiments of the invention and the Examples included here. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as normally understood by those skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain preferred methods and materials are described here. In describing the modalities and claim of the invention, certain terminology will be used according to the definitions provided below.
[00046] As used here, the singular forms "one", "one", "0" and "a" include references in the plural, unless otherwise indicated. Thus, for example, references to the "method" include one or more methods and / or steps of the type described here and references to "an eTEC spacer" refer to one or more eTEC spacers, as will be evident to those skilled in the art when reading the description.
[00047] As used herein, the term "about" means within a statistically significant range of a value, such as a range of concentration, time period, molecular weight, temperature or pH provided. Such a range may be within an order of magnitude, typically within 20%, more typically within 10% and, even more typically, within 5% of the indicated value or range. Sometimes, such a range may be within the experimental error typical of conventional methods used for measuring and / or determining a given value or range. The permitted variation covered by the term "about" will depend on the system under study in particular and can be readily appreciated by one of those skilled in the art. Whenever a range is cited in the present application, each integer within the range is also considered as an embodiment of the invention.
[00048] It will be noted that, in the present description, terms such as "comprises", "understood", "comprising", "contains", "containing" and the like may have the meaning ascribed to them in the United States patent law ; for example, they can mean "includes", "included", "including", and so on. Such terms refer to the inclusion of ingredients or a set of particular ingredients without excluding any other ingredients. Terms such as "consisting essentially of" and "consisting essentially of" have the meaning ascribed to them in United States patent law, for example, they allow the inclusion of additional ingredients or steps that do not prejudice the new features or basic principles of the invention, that is, they exclude additional ingredients or unmentioned steps that impair the new or basic characteristics of the invention. The terms "consisting of" and "consists of" have the meaning ascribed to them in United States patent law; that is, these terms have a closed meaning. Consequently, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.
[00049] The term "saccharide", as used here, can refer to a polysaccharide, an oligosaccharide or a monosaccharide. Frequently, references to a saccharide refer to a bacterial capsular polysaccharide, in particular capsular polysaccharides derived from pathogenic bacteria, such as S. pneumoniae or N. meningitidis.
[00050] The terms "conjugate" or "glycoconjugate" are used interchangeably here to refer to a saccharide covalently conjugated to a carrier protein. The glycoconjugates of the present invention are sometimes referred to herein as "eTEC-linked" glycoconjugates, which comprise a saccharide covalently conjugated to a carrier protein by means of at least one eTEC spacer. The glycoconjugates bound by eTEC of the invention and immunogenic compositions comprising them may contain a certain amount of free saccharide.
[00051] The term "free saccharide", as used here, means a saccharide that is not covalently conjugated to the carrier protein or a saccharide that is covalently bound to very few carrier proteins joined in a high ratio of saccharide / protein (> 5: 1) but is nevertheless present in the glycoconjugate composition. The free saccharide may not be covalently associated (i.e., not covalently bound, adsorbed to or terminated in or with) the conjugated saccharide-carrier protein glycoconjugate. The terms "free polysaccharide" and "free capsular polysaccharide" can be used here to convey the same meaning with respect to glycoconjugates in which the saccharide is a polysaccharide or a capsular polysaccharide, respectively.
[00052] As used herein, "conjugate", "conjugate" and "conjugation" refer to a process by which a saccharide, for example, a bacterial capsular polysaccharide, is covalently linked to a carrier molecule or carrier protein. In the methods of the present invention, saccharide is covalently conjugated to the carrier protein through at least one eTEC spacer. The conjugation can be carried out according to the methods described below or other processes known in the art. Conjugation to a carrier protein increases the immunogenicity of a bacterial capsular polysaccharide. Glycoconjugates
[00053] The present invention relates to glycoconjugates which comprise a saccharide covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate bond and where the protein The carrier is covalently coupled to the eTEC spacer via an amide bond.
[00054] In addition to the presence of one or more eTEC spacers, new characteristics of the glycoconjugates of the present invention include the resulting molecular weight profiles of the resulting eTEC-linked saccharides and glycoconjugates, the proportion of conjugated lysines per carrier protein and the number of covalently lysines linked to the polysaccharide via the eTEC spacer (s), 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 the total amount of saccharide.
[00055] The eTEC-linked glycoconjugates of the invention can be represented by the general formula (I):

[00056] The eTEC spacer includes seven linear atoms (i.e., - C (O) NH (CH2) 2SCH2C (O) -) and provides stable thioether and amide bonds between the carrier protein and the saccharide. Synthesis of the eTEC-linked glycoconjugate involves reaction of an activated hydroxyl group of the saccharide with the amino group of a thioalkylamine reagent, for example, cystamine or cystinamine or a salt thereof, formation of a carbamate bond to the saccharide to provide use a thiolated saccharide. Generation of one or more free sulfhydryl groups is carried out by reaction with a reducing agent to provide an activated thiolated saccharide. Reaction of the free sulfhydryl groups of the activated thiolated saccharide with an activated carrier protein having one or more α-haloacetamide groups in residues containing amine generates a thioether bond to form the conjugate, in which the carrier protein is linked to the eTEC spacer via an amide bond.
[00057] In glycoconjugates of the invention, the saccharide can be a polysaccharide, an oligosaccharide or a monosaccharide and the carrier protein can be selected from any suitable vehicle, as further described herein or known to those skilled in the art. In frequent modalities, saccharide is a bacterial capsular polysaccharide. In some of these modalities, the carrier protein is CRM197.
[00058] In some of such embodiments, the eTEC-linked glycoconjugate comprises a S. pneumoniae-derived capsular polysaccharide Pn. In specific embodiments, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In other embodiments, the capsular polysaccharide is selected from the group consisting of capsular polysaccharides Pn-serotypes 10A, 11 A, 22F and 33F. In one of such embodiments, the capsular polysaccharide is a Pn-33F capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Pn-22F capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a Pn-10A capsular polysaccharide. In yet another such embodiment, the capsular polysaccharide is a Pn-11A capsular polysaccharide.
[00059] In other embodiments, the eTEC-linked glycoconjugate comprises a capsular polysaccharide Mn derived from / V. meningitidis. In specific embodiments, the capsular polysaccharide Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y. In one of these modalities, the capsular polysaccharide is a capsular polysaccharide Mn-A. In another such embodiment, the capsular polysaccharide is an Mn-C capsular polysaccharide. In another such embodiment, the capsular polysaccharide is a capsular polysaccharide Mn-W135. In yet another such embodiment, the capsular polysaccharide is an Mn-Y capsular polysaccharide.
[00060] In particularly preferred embodiments, the eTEC-linked glycoconjugate comprises a bacterial capsular polysaccharide Pn or Mn, such as a capsular polysaccharide Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F or an Mn-serotype A, C, W135 or Y capsular polysaccharide, which is covalently conjugated to CRM197 using an eTEC spacer.
[00061] In some embodiments, the eTEC-linked glycoconjugates of the present invention comprise a saccharide covalently conjugated to the carrier protein by means of an eTEC spacer, wherein the saccharide has a molecular weight between 10 kDa and 2,000 kDa. In other embodiments, the saccharide has a molecular weight between 50 kDa and 2,000 kDa. In other such embodiments, the saccharide has a molecular weight between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500 kDa. In some of these embodiments, saccharide is a bacterial capsular polysaccharide, such as a Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B capsular polysaccharide , 18C, 19A, 19F, 22F, 23F or 33F or an Mn-serotype A, C, W135 or Y capsular polysaccharide, wherein the capsular polysaccharide has a molecular weight that falls within any of the molecular weight ranges as described.
[00062] In some embodiments, the eTEC-linked glycoconjugate of the present invention has a molecular weight between 50 kDa and 20000 kDa. In other embodiments, eTEC-linked glycoconjugate has a molecular weight between 500 kDa and 10,000 kDa. In other embodiments, the eTEC-linked glycoconjugate has a molecular weight between 200 kDa and 10,000 kDa. In still other embodiments, the eTEC-linked glycoconjugate has a molecular weight between 1,000 kDa and 3000 kDa.
[00063] In other embodiments, the eTEC-linked glycoconjugate of the present invention has a molecular weight between 200 kDa and 20,000 kDa; between 200 kDa and 15,000 kDa; between 200 kDa and 10,000 kDa; between 200 kDa and 7,500 kDa; between 200 kDa and 5,000 kDa; between 200 kDa and 3,000 kDa; between 200 kDa and 1,000 kDa; between 500 kDa and 20,000 kDa; between 500 kDa and 15,000 kDa; between 500 kDa and 12,500 kDa; between 500 kDa and 10,000 kDa; between 500 kDa and 7,500 kDa; between 500 kDa and 6,000 kDa; between 500 kDa and 5,000 kDa; between 500 kDa and 4000 kDa; between 500 kDa and 3000 kDa; between 500 kDa and 2,000 kDa; between 500 kDa and 1,500 kDa; between 500 kDa and 1,000 kDa; between 750 kDa and 20,000 kDa; 750 kDa and 15,000 kDa; between 750 kDa and 12,500 kDa; between 750 kDa and 10,000 kDa; between 750 kDa and 7,500 kDa; between 750 kDa and 6,000 kDa; between 750 kDa and 5,000 kDa; between 750 kDa and 4,000 kDa; between 750 kDa and 3000 kDa; between 750 kDa and 2,000 kDa; between 750 kDa and 1,500 kDa; between 1,000 kDa and 15,000 kDa; between 1,000 kDa and 12,500 kDa; between 1,000 kDa and 10,000 kDa; between 1,000 kDa and 7,500 kDa; between 1,000 kDa and 6,000 kDa; between 1,000 kDa and 5,000 kDa; between 1,000 kDa and 4,000 kDa; between 1,000 kDa and 2,500 kDa; between 2,000 kDa and 15,000 kDa; between 2,000 kDa and 12,500 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 6,000 kDa; between 2,000 kDa and 5,000 kDa; between 2,000 kDa and 4,000 kDa; or between 2,000 kDa and 3,000 kDa.
[00064] Another way to characterize the eTEC-linked glycoconjugates of the invention is through the number of lysine residues in the carrier protein that become conjugated to the saccharide through an eTEC spacer, which can be characterized as a band of conjugated lysines.
[00065] In frequent embodiments, the carrier protein is covalently conjugated to the eTEC spacer through an amide bond to one or more a-amino groups of lysine residues on the carrier protein. In some of such embodiments, the carrier protein comprises 2 to 20 lysine residues covalently conjugated to the saccharide. In other embodiments, the carrier protein comprises 4 to 16 lysine residues covalently conjugated to the saccharide.
[00066] In a preferred embodiment, the carrier protein comprises CRM197, which contains 39 lysine residues. In some of such embodiments, CRM197 may comprise 4 to 16 lysine residues from 39 covalently linked to saccharide. Another way of expressing this parameter is that about 10% to about 41% of lysines in CRM197 are covalently linked to saccharide. In another such embodiment, CRM197 may comprise 2 to 20 lysine residues from 39 covalently bound to the saccharide. Another way of expressing this parameter is that about 5% to about 50% of CRM197's lysines are covalently bound to saccharide.
[00067] The eTEC-linked glycoconjugates of the invention can also be characterized by the ratio (w / w) of saccharide to carrier protein. In some embodiments, the saccharide: carrier protein (w / w) ratio is between 0.2 and 4. In other embodiments, the saccharide: carrier protein (w / w) ratio is between 1.0 and 2.5. In other modalities, the saccharide: carrier protein (w / w) ratio is between 0.4 and 1.7. In some of these embodiments, saccharide is a bacterial capsular polysaccharide and / or the carrier protein is CRM197.
[00068] Glycoconjugates can also be characterized by the number of covalent bonds between the carrier protein and the saccharide as a function of saccharide repeat units. In one embodiment, the glycoconjugate of the invention comprises at least one covalent bond between the carrier protein and the polysaccharide for every 4 saccharide repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeating units of the polysaccharide. In another fashion, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeat units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeating units of the polysaccharide.
[00069] In frequent embodiments, the carrier protein is CRM197 and the covalent bond through an eTEC spacer between CRM197 and the polysaccharide occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide.
[00070] An important consideration during conjugation is the development of conditions that allow the retention of potentially sensitive non-saccharide substituting functional groups of individual components, such as side chains of O-acyl, phosphate or glycerol phosphate, which may form part of the epitope saccharide.
[00071] In one embodiment, the glycoconjugate comprises a saccharide that has a degree of O-acetylation between 10-100%. In some of these embodiments, the saccharide has a degree of O-acetylation between 50-100%. In other embodiments, the saccharide has a degree of O-acetylation between 75-100%. In other embodiments, saccharide has an O-acetylation degree greater than or equal to 70% (> 70%).
[00072] Glycoconjugates bound by eTEC and immunogenic compositions of the invention may contain free saccharide that is not covalently conjugated to the carrier protein but is nevertheless present in the glycoconjugate composition. The free saccharide may not be covalently associated (i.e., not covalently bound, adsorbed to or terminated in or with) the glycoconjugate.
[00073] In some embodiments, eTEC-linked glycoconjugate comprises less than about 45% free saccharide, less than about 40% free saccharide, less than about 35% free saccharide, less than about 30 % free saccharide, less than about 25% free saccharide, less than about 20% free saccharide, less than about 15% free saccharide, less than about 10% free saccharide or less than about 5 % free saccharide in relation to the total amount of saccharide. Preferably, the glycoconjugate comprises less than 15% free saccharide, more preferably less than 10% free saccharide and, even more preferably, less than 5% free saccharide.
[00074] In certain preferred embodiments, the invention provides an eTEC-linked glycoconjugate comprising a capsular polysaccharide, preferably a Pn or Mn capsular polysaccharide, covalently conjugated to a carrier protein by means of an eTEC spacer having one or more of the following characteristics , alone or in combination: the polysaccharide has a molecular weight between 50 kDa and 2,000 kDa; glycoconjugate has a molecular weight between 500 kDa to 10,000 kDa; the carrier protein comprises 2 to 20 lysine residues covalently linked to the saccharide; the saccharide: carrier protein (w / w) ratio is between 0.2 and 4; the glycoconjugate comprises at least one covalent bond between the carrier protein and the polysaccharide for every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide; the saccharide has a degree of O-acetylation between 75-100%; the conjugate comprises less than about 15% free polysaccharide in relation to the total polysaccharide; the carrier protein is CRM197; The capsular polysaccharide is selected from capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F or 0 capsular polysaccharide is selected from Mn-serotype A, C, W135 or Y capsular polysaccharides.
[00075] Glycoconjugates bound by eTEC can also be characterized by their molecular size distribution (Kd). The molecular size of the conjugates is determined by size exclusion chromatography (SEC - Size Exclusion Chromatography) in a stationary phase in Sepharose CL-4B medium using a high pressure liquid chromatography system (High Pressure Liquid Chromatography - HPLC) . For Kd determination, the chromatography column is first calibrated to determine Vo, which represents the void volume or total exclusion volume, and Vi, the volume at which the smallest molecules in the sample elute, which is also known as the interparticle volume. Every SEC separation occurs between Vo and V. The Kd value for each fraction collected is determined by the following expression: Kd = (Ve-Vi) / (Vi-V0), where Ve represents the volume of retention of the compound. The fraction% (main peak) that elutes <0.3 defines the Kd (molecular size distribution) of the conjugate. In some embodiments, the invention provides eTEC-linked glycoconjugates having a molecular size distribution (Kd) of> 35%. In other embodiments, the invention provides eTEC-linked glycoconjugates having a molecular size distribution (Kd) of> 15%,> 20%,> 25%,> 30%,> 35%,> 40%,> 45%,> 50%,> 60%,> 70%,> 80% or> 90%.
[00076] The glycoconjugates bound by eTEC and immunogenic compositions of the invention may contain free sulfhydryl residues. In some cases, activated thiolated saccharides formed by the methods provided here will contain multiple free sulfhydryl residues, some of which may not undergo covalent conjugation with the carrier protein during the conjugation step. Such residual free sulfhydryl residues are "capped" by reaction with a thiol-reactive "capping" reagent, for example, iodoacetamide (IAA), for "capping" of potentially reactive functionality. Other thiol-reactive "capping" reagents, for example, reagents containing maleimide and so on, are also considered.
[00077] In addition, eTEC-linked glycoconjugates and immunogenic compositions of the invention may contain residual unconjugated carrier protein, which may include activated carrier protein that has undergone modification during the steps of the "capping" process.
[00078] The glycoconjugates of the invention can be used in the production of immunogenic compositions to protect receptors against bacterial infections, for example, by pathogenic bacteria, such as S. pneumoniae or N. meningitidis. Thus, in another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a saccharide covalently conjugated to a carrier protein by means of an eTEC spacer. , as described here.
[00079] In frequent embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide.
[00080] In some of these embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate which comprises a S. pneumoniae-derived capsular polysaccharide. In some specific embodiments, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.
[00081] In other such embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate which comprises an Mn capsular polysaccharide derived from N. meningitidis. In some specific embodiments, the capsular polysaccharide Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y.
[00082] In particularly preferred embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate which comprises a bacterial capsular polysaccharide, such as a Pn or Mn capsular polysaccharide, covalently conjugated to CRM197 by means of an eTEC spacer.
[00083] In some embodiments, the immunogenic composition comprises an adjuvant. In some of these 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.
[00084] The eTEC-linked glycoconjugates of the invention and immunogenic compositions comprising them may contain a certain amount of free saccharide. In some embodiments, the immunogenic composition comprises less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10% or less than about 5% of free polysaccharide compared to the total amount of polysaccharide. Preferably, the immunogenic composition comprises less than 15% free saccharide, more preferably less than 10% free saccharide and, even more preferably, less than 5% free saccharide.
[00085] In another aspect, the glycoconjugates or immunogenic compositions of the invention can be used to generate antibodies that are functional, as measured by the death of bacteria in an animal efficacy model or by means of an opsonophagocytic death assay. Glycoconjugates of the invention comprising a bacterial capsular polysaccharide can be used in the production of antibodies against such a bacterial capsular polysaccharide. Such antibodies can subsequently be used in research and clinical laboratory tests, such as bacterial detection and serotyping. Such antibodies can also be used to confer passive immunity on an individual. In some embodiments, antibodies produced against bacterial polysaccharides are functional in an animal efficacy model or in an opsonophagocytic death assay.
[00086] The glycoconjugates bound by eTEC and immunogenic compositions described here can also be used in various therapeutic or prophylactic methods for preventing, treating or ameliorating an infection, disease or bacterial condition in an individual. In particular, eTEC-linked glycoconjugates comprising a bacterial antigen, such as a bacterial capsular polysaccharide from a pathogenic bacterium, can be used to prevent, treat or ameliorate an infection, disease or bacterial condition in an individual caused by pathogenic bacteria.
[00087] Thus, in one aspect, the invention provides a method of preventing, treating or ameliorating an infection, disease or bacterial condition in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition of the invention, in that said immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide.
[00088] In one embodiment, the infection, disease or condition is associated with S. pneumoniae bacteria and the glycoconjugate comprises a capsular polysaccharide Pn. In some of these modalities, the infection, disease or condition is selected from the group consisting of pneumonia, sinusitis, otitis media, meningitis, bacteremia, septicemia, pleural emphysema, conjunctivitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, mastoiditis, cellulitis, soft tissue infection and brain abscess.
[00089] In another embodiment, the infection, disease or condition is associated with bacteria A /, meningitidis and the glycoconjugate comprises a capsular polysaccharide Mn. In some of these modalities, the infection, disease or condition is selected from the group consisting of meningitis, meningococcemia, bacteremia and septicemia.
[00090] In another aspect, the invention provides a method of inducing an immune response in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically-excipient, carrier or diluent acceptable, wherein the glycoconjugate comprises a bacterial capsular polysaccharide.
[00091] In yet another aspect, the invention provides a method for preventing, treating or ameliorating a disease or condition caused by pathogenic bacteria in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition comprising a bound glycoconjugate by eTEC and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide.
[00092] In another aspect, the invention provides a method for reducing the severity of at least one symptom of a disease or condition caused by an infection by pathogenic bacteria comprising administering to an individual an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide, for example, a Pn or Mn capsular polysaccharide.
[00093] In another aspect, the invention provides a method of administering an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate of the invention to an individual to generate a protective immune response in the individual, as further described herein.
[00094] In yet another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate of the present invention, as described herein, for use in preventing, treating or ameliorating a bacterial infection, for example, an infection by S. pneumoniae or N. meningitidis.
[00095] In another aspect, the invention provides the use of an immunogenic composition comprising an eTEC-linked glycoconjugate of the present invention, as described herein, for the preparation of a medicament for the prevention, treatment or amelioration of a bacterial infection, for example , infection by S. pneumoniae or N. meningitidis.
[00096] In the therapeutic and / or prophylactic methods and uses described above, the immunogenic composition often comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide covalently linked to a carrier protein by means of an eTEC spacer. In frequent embodiments of the methods described here, the bacterial capsular polysaccharide is a Pn capsular polysaccharide or an Mn capsular polysaccharide. In some of these modalities, the capsular polysaccharide is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C , 19A, 19F, 22F, 23F and 33F. In other embodiments, the capsular polysaccharide is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y.
[00097] In certain preferred embodiments, the carrier protein is CRM197. In particularly preferred embodiments, the immunogenic composition comprises an eTEC-linked glycoconjugate comprising a bacterial capsular polysaccharide, such as a Pn or Mn capsular polysaccharide, covalently attached to CRM197 by means of an eTEC spacer.
[00098] In addition, the present invention provides methods for inducing an immune response against S. pneumoniae or N. meningitidis bacteria in an individual, methods for preventing, treating or ameliorating an infection, disease or condition caused by S. pneumoniae bacteria or N. meningitidis in an individual and methods for reducing the severity of at least one symptom of an infection, disease or condition caused by an infection by S. pneumoniae or N. meningitidis in an individual, in each case through administration, to the individual , of an immunologically effective amount of an immunogenic composition comprising an eTEC-linked glycoconjugate and a pharmaceutically acceptable excipient, vehicle or diluent, wherein the glycoconjugate comprises a bacterial capsular polysaccharide derived from S. pneumoniae or N. meningitidis, respectively. Saccharides
[00099] Saccharides can include polysaccharides, oligosaccharides and monosaccharides. In frequent embodiments, saccharide is a polysaccharide, in particular a bacterial capsular polysaccharide. Capsular polysaccharides are prepared by conventional techniques known to those skilled in the art.
[000100] In the present invention, capsular polysaccharides can be prepared, for example, from Pn-serotypes 1,3,4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F of S. pneumoniae. In one embodiment, each pneumococcal polysaccharide serotype can be grown in a soy-based medium. Individual polysaccharides are purified by centrifugation, precipitation, ultrafiltration and / or column chromatography. Purified polysaccharides can be activated to make them capable of reacting with the eTEC spacer and then incorporated into the glycoconjugates of the invention, as further described here.
[000101] The molecular weight of the capsular polysaccharide is a consideration for use in immunogenic compositions. High molecular weight capsular polysaccharides 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 high molecular weight capsular polysaccharides are considered for use in the conjugates, compositions and methods of the present invention.
[000102] In some embodiments, saccharide has a molecular weight between 10 kDa and 2,000 kDa. In other embodiments, the saccharide has a molecular weight between 50 kDa and 2,000 kDa. In other such embodiments, the saccharide has a molecular weight between 50 kDa and 1,750 kDa; between 50 kDa and 1,500 kDa; between 50 kDa and 1,250 kDa; between 50 kDa and 1,000 kDa; between 50 kDa and 750 kDa; between 50 kDa and 500 kDa; between 100 kDa and 2,000 kDa; between 100 kDa and 1,750 kDa; between 100 kDa and 1,500 kDa; between 100 kDa and 1,250 kDa; between 100 kDa and 1,000 kDa; between 100 kDa and 750 kDa; between 100 kDa and 500 kDa; between 200 kDa and 2,000 kDa; between 200 kDa and 1,750 kDa; between 200 kDa and 1,500 kDa; between 200 kDa and 1,250 kDa; between 200 kDa and 1,000 kDa; between 200 kDa and 750 kDa; or between 200 kDa and 500 kDa. In some of these embodiments, saccharide is a bacterial capsular polysaccharide, such as a Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B capsular polysaccharide , 18C, 19A, 19F, 22F, 23F or 33F or an Mn-serotype A, C, W135 or Y capsular polysaccharide, wherein the capsular polysaccharide has a molecular weight that falls within one of the molecular weight ranges as described .
[000103] In some embodiments, the saccharides of the invention are O-acetylated. In some embodiments, the glycoconjugate comprises a saccharide that has a degree of O acetylation between 10-100%, between 20 to 100%, between 30-100%, between 40-100%, between 50-100%, between 60- 100%, between 70-100%, between 75-100%, 80-100%, 90-100%, 50- 90%, 60-90%, 70-90% or 80-90%. In other embodiments, the degree of O-acetylation is> 10%, 20%>,> 30%, 40%>,> 50%, 60%>,> 70%,> 80% or> 90% or about 100 %.
[000104] In some embodiments, the capsular polysaccharides, glycoconjugates or immunogenic compositions of the invention are used to generate antibodies that are functional, as measured by the death of bacteria in an animal efficacy model or an opsonophagocytic death assay that demonstrates that antibodies kill bacteria.
[000105] Capsular polysaccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art. See, for example, Fournier et al .. (1984), supra; Fournier et al. (1987) Ann. Inst. Pasteur / Microbiol. 138: 561 567; United States Patent Application Publication No. 2007/0141077; and International Patent Application Publication No. WO 00/56357; each of which is incorporated herein by reference as if presented in full). In addition, they can be produced using synthesis protocols. In addition, the capsular polysaccharide can be produced recombinantly using genetic engineering procedures also known to those skilled in the art (see Sau et al. (1997) Microbiology 143: 2395 2405; and United States Patent No. 6,027,925; each of which is incorporated herein by reference as if presented in full).
[000106] The bacterial strains of S. pneumoniae or N. meningitidis used to make the respective polysaccharides that are used in the glycoconjugates of the invention can be obtained from collections of established cultures or from clinical specimens. Vehicle Proteins
[000107] Another component of the glycoconjugate of the present invention is a carrier protein to which the saccharide is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" can be used interchangeably here. Carrier proteins are preferably proteins that are non-toxic and non-reactogenic and obtained in sufficient quantity and purity. Vehicle proteins should be subject to conventional conjugation procedures. In the novel glycoconjugates of the invention, the carrier protein is covalently linked to a saccharide via an eTEC spacer.
[000108] Conjugation to a vehicle can increase the immunogenicity of an antigen, for example, bacterial antigen, such as a bacterial capsular polysaccharide. Preferred protein vehicles for antigens are toxins, toxoids or any mutant cross-reactive material (Cross-Reactive Material - CRM) for tetanus toxin, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus and Streptococcus. In one embodiment, a particularly preferred vehicle is the CRM197 diphtheria toxoid, derived from the C. diphtheriae strain C7 (β197), which produces the CRM197 protein. This strain has ATCC Accession No. 53281. A method for producing CRM197 is described in United States Patent No. 5,614,382, which is incorporated herein by reference as if presented in its entirety.
[000109] Alternatively, a fragment or epitope of the carrier protein or other 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. See United States Patent Application No. 150,688, filed on February 1, 1988, entitled "Synthetic Peptides Representing a T-Cell Epi-tope as a Carrier Molecule For Conjugate Vaccines"; incorporated herein by reference as if presented in full. Other suitable carrier proteins include inactivated bacterial toxins, such as the cholera toxoid (for example, as described in International Patent Application WO 2004/083251), E. coli LT, E. coli ST and exotoxin A of Pseudomonas aeruginosa. Bacterial outer membrane proteins, such as outer membrane complex C (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA) or Haemophilus protein D influenzae, can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (Bovine Serum Albumin - BSA) or tuberculin purified protein derivative (PPD), can also be used as proteins vehicle.
[000110] Consequently, in frequent modalities, eTEC-linked glycoconjugates comprise CRM197 as a carrier protein, in which the capsular polysaccharide is covalently linked to the eTEC spacer via a carbamate bond and in which CRM197 is covalently linked to the eTEC spacer via an amide bond formed by an activated amino acid residue of the protein, typically through the α-amine group of one or more lysine residues.
[000111] 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 some embodiments of the immunogenic compositions, CRM197 can comprise from 4 to 16 lysine residues from 39 covalently bound to the saccharide. Another way of expressing this parameter is that about 10% to about 41% of the lysines in CRM197 are covalently linked to saccharide. In other embodiments, CRM197 may comprise 2 to 20 lysine residues from 39 covalently linked to saccharide. Another way of expressing this parameter is that about 5% to about 50% of the lysines in CRM197 are covalently linked to saccharide.
[000112] The frequency of binding of the saccharide chain to a lysine in the carrier protein is another parameter for characterizing the glycoconjugates of the invention. For example, in some embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 4 saccharide repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 10 saccharide repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 15 saccharide repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once in every 25 saccharide repeating units of the polysaccharide.
[000113] In frequent embodiments, the carrier protein is CRM197 and the covalent bond through an eTEC spacer between CRM197 and the polysaccharide occurs at least once in every 4, 10, 15 or 25 saccharide repeat units of the polysaccharide. In some of these embodiments, the polysaccharide is a bacterial capsular polysaccharide derived from S. pneumoniae or N. meningitidis.
[000114] In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for each 5 to 10 saccharide repeating units; every 2 to 7 saccharide repeat units; every 3 to 8 saccharide repetition units; every 4 to 9 saccharide repeat units; every 6 to 11 saccharide repeat units; every 7 to 12 saccharide repeat units; every 8 to 13 saccharide repeat units; every 9 to 14 saccharide repetition units; every 10 to 15 saccharide repetition units; every 2 to 6 saccharide repeat units, every 3 to 7 saccharide repeat units; every 4 to 8 saccharide repeat units; every 6 to 10 saccharide repeat units; every 7 to 11 saccharide repeat units; every 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 repeating units; or every 4 to 25 saccharide repeat units.
[000115] 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 saccharide repeat units of the polysaccharide. Methods for Production of eTEC-Linked Glycoconjugates
[000116] The present invention provides methods for preparing eTEC-linked glycoconjugates which comprise a saccharide covalently conjugated to a carrier protein by means of a (2 - ((2-oxoethyl) thio) ethyl) carbamate spacer (ETEC). The eTEC spacer contains seven linear atoms (i.e., -C (O) NH (CH2) 2SCH2C (O) -), comprising stable thioether and amide bonds and serves to covalently link saccharide and carrier protein. One end of the eTEC spacer is covalently linked to a hydroxyl group of the saccharide via a carbamate bond. The other end of the eTEC spacer is covalently attached to the amino-containing residue of the carrier protein, typically an α-lysine residue, via an amide bond.
[000117] A representative pathway for the preparation of glycoconjugates of the present invention comprising a polysaccharide conjugated to the activated carrier protein CRM197 is shown in Figure 1. The chemical structure of a representative bacterial capsular polysaccharide, the pneumococcal polysaccharides of serotypes 33F, 10A, 11A and 22F derived from S. pneumoniae having potential sites of modification using the eTEC spacer process are shown in Figure 2, Figure 3, Figure 4 and Figure 5, respectively.
[000118] The structure of an eTEC-linked glycoconjugate representative of the present invention comprising the serotype 33F pneumococcal polysaccharide covalently conjugated to CRM197 using the eTEC ligand chemistry is shown in Figure 6 (A). Potential free sulfhydryl sites with "cap" and without "cap" are shown in Figure 6 (B) for illustrative purposes. Polysaccharides usually contain multiple hydroxyl groups and the binding site of the eTEC spacer to a specific hydroxyl within the polysaccharide repeating units through the carbamate bond, therefore, it can vary.
[000119] In one aspect, the method comprises the steps of: a) reacting a saccharide with a carbonic acid derivative, such as 1,1'-carbonyl-di- (1,2,4-triazole) (CDT) or 1, T-carbonyldiimidazole (CDI), in an organic solvent to produce an activated saccharide; b) reaction of the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reaction of the thiolated saccharide-carrier protein conjugate with (i) a first "capping" reagent capable of "capping" unconjugated α-haloacetamide groups from the activated carrier protein; and / or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide; wherein an eTEC-linked glycoconjugate is produced.
[000120] In a particularly preferred embodiment, the method comprises the steps of: a) reacting a Pn-33F capsular polysaccharide with CDI or CDT in an organic solvent to produce an activated Pn 33F polysaccharide; b) reaction of the activated Pn-33F polysaccharide with cystamine or cysteinamine a salt thereof to produce a thiolated Pn-33F polysaccharide; c) reacting the thiolated Pn-33F polysaccharide with a reducing agent to produce a thiolated activated Pn-33F polysaccharide comprising one or more free sulfhydryl residues; d) reaction of the thiolated activated Pn-33F polysaccharide with a CRM197 activated carrier protein comprising one or more α-bromoacetamide groups to produce a thiolated PN-33F-CRMw polysaccharide conjugate; and e) reaction of the thiolated PN-33F polysaccharide conjugate-CRMi97 with (i) N-acetyl-L-cysteine as a first capping reagent capable of "capping" unconjugated o-bromoacetamide groups of the activated carrier protein; and (ii) iodoacetamide as a second capping reagent capable of capping unconjugated free sulfhydryl residues of the thiolated activated Pn-33F polysaccharide; wherein an eTEC-linked Pn-33F-CRMi97 polysaccharide glycoconjugate is produced.
[000121] In frequent modalities, the derivative of carbonic acid is CDT or CDI. Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). Freeze-drying of the activated saccharide is not necessary before the thiolation and / or conjugation steps.
[000122] In a preferred embodiment, the thiolated saccharide is produced by reacting the activated saccharide with the symmetrical bifunctional reagent of thioalkylamine cystamine or a salt thereof. A potential advantage for this reagent is that the symmetrical cischarin linker can react with two molecules of activated saccharide, thus forming two molecules of thiolated saccharide per molecule of cystamine when reducing the disulfide bond. Alternatively, the thiolated saccharide can be formed by reacting the activated saccharide with cysteamine or a salt thereof. The glycoconjugates linked by eTEC produced by the methods of the invention can be represented by the general formula (I).
[000123] It will be understood by those skilled in the art that step c) is optional when the activated saccharide is reacted with cysteamine or a salt thereof containing free sulfhydryl residues. As a practical matter, thiolated saccharides comprising cysteamine are routinely reacted with a reducing agent in step c) to reduce any oxidized disulfide by-products that may be formed during the reaction.
[000124] In some embodiments of this aspect, step d) further comprises providing an activated carrier protein comprising one or more α-haloacetamide groups, prior to reaction of the activated thiolated saccharide with the activated carrier protein to produce a thiolated-saccharide conjugate carrier protein. In frequent embodiments, the activated carrier protein comprises one or more a-bromoacetamide groups.
[000125] The thiolated saccharide-carrier protein conjugate can be treated with one or more "capping" reagents capable of reacting with residual activated functional groups present in the reaction mixture. Such residual reactive groups may be present in unreacted components of saccharides or carrier protein due to incomplete conjugation or the presence of an excess of one of the components in the reaction mixture. In this case, "capping" can assist in the purification or isolation of glycoconjugate. In some cases, residual activated functional groups may be present in the glycoconjugate.
[000126] For example, excess α-haloacetamide groups in the activated carrier protein can be "capped" by reaction with a low molecular weight thiol, such as N-acetyl-L-cysteine, which can be used in excess to ensure complete capping. "Capping" with N-acetyl-L-cysteine also allows confirmation of the conjugation efficiency by detecting the single amino acid S-carboxymethylcysteine (CMC) of the cysteine residues in the "cap" sites, which can be determined by acid hydrolysis and analysis amino acids in the conjugation products. Detection of this amino acid confirms successful "capping" of the reactive bromoacetamide groups, thus making them unavailable for any undesirable chemical reactions. Acceptable levels of covalence and capping are between about 1-15 for CMCA / Lys and about 0-5 for CMC / Lys. Similarly, excess free sulfhydryl residues can be "capped" by reaction with a low molecular weight electrophilic reagent, such as iodacetamide. A part of the CMCA can be derived from the capped polysaccharide thiols directly by iodine-acetamide that was not involved in conjunction with the haloacyl groups of the carrier protein. Therefore, post-conjugation reaction samples (prior to iodoacetamide capping) need to be examined by amino acid analysis (CMCA) to determine the exact levels of thiols directly involved in the conjugation. For a thiolated saccharide containing 10-12 thiols it is determined that, typically, 5-6 thiols are directly involved in the conjugation between the polysaccharide thiol and bromoacetylated protein and 4-5 thiols are capped by iodoacetamide.
[000127] In preferred embodiments, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamide groups on the carrier protein. In other embodiments, the second capping reagent is iodoacetamide (IAA), which reacts with free, unconjugated sulfhydryl groups of the activated thiolated saccharide. Frequently, step e) comprises capping with N-acetyl-L-cysteine as the first capping reagent and IAA as the second capping reagent. In some modalities, the "capping" step e) further comprises reaction with a reducing agent, for example, DTT, TCEP or mercaptoethanol, after reaction with the first and / or second "capping" reagents.
[000128] In some embodiments, the method further comprises a step of purifying the glycoconjugate bound by eTEC, for example, by means of ultrafiltration / diafiltration.
[000129] In a preferred embodiment, the symmetrical bifunctional reagent of thioalkylamine is cystamine or a salt thereof, which is reacted with the activated saccharide to provide a thiolated saccharide or a salt thereof containing a disulfide portion.
[000130] Reaction of such thiolated saccharide derivatives with a reducing agent produces an activated thiolated polysaccharide comprising one or more free sulfhydryl residues. Such activated thiolated saccharides can be isolated and purified, for example, by means of ultrafiltration / diafiltration. Alternatively, activated thiolated saccharides can be isolated and purified, for example, by conventional size exclusion chromatography (SEC) methods or ion exchange chromatographic methods, such as DEAE, known in the art.
[000131] In the case of cystamine-derived thiolated saccharides, reaction with a reducing agent cleaves the disulfide bond to provide an activated thiolated saccharide comprising one or more free sulfhydryl residues. In the case of thiolated saccharides derived from cysteamine, reaction with a reducing agent is optional and can be used to reduce disulfide bonds formed by oxidation of the reagent or product.
[000132] Reducing agents used in the methods of the invention include, for example, tris (2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT) or mercaptoethanol. However, any suitable disulfide reducing agent can be used.
[000133] In some embodiments, the methods further comprise providing an activated carrier protein comprising one or more α-haloacetamide groups, preferably one or more α-bromoacetamide groups.
[000134] Reaction of the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide portions results in nucleophilic displacement of the α-halo group of the activated carrier protein by one or more free sulfhydryl groups of the activated thiolated saccharide, forming the bond of thioether of the eTEC spacer.
[000135] The α-haloacetylated amino acid residues of the carrier protein are typically linked to the α-amino groups of one or more lysine residues of the carrier protein. In frequent embodiments, the carrier protein contains one or more α-bromoacetylated amino acid residues. In one embodiment, the carrier protein is activated with a bromoacetic acid reagent, such as the bromoacetic acid N-hydroxysuccinimide ester (BAANS).
[000136] In one embodiment, the method includes the step of providing an activated carrier protein comprising one or more α-haloacetamide groups and reacting the activated thiolated polysaccharide with the activated carrier protein to produce a thiolated polysaccharide-carrier protein conjugate by that a glycoconjugate comprising a polysaccharide conjugated to a carrier protein by means of an eTEC spacer is produced.
[000137] In some preferred embodiments of the methods included here, the bacterial capsular polysaccharide is a Pn capsular polysaccharide derived from S. pneumoniae. In some of these modalities, the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F. In certain preferred embodiments, the carrier protein is CRM197 and the capsular polysaccharide Pn is selected from the group consisting of capsular polysaccharides Pn-serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A , 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.
[000138] In other preferred embodiments of the methods provided here, the bacterial capsular polysaccharide is an Mn capsular polysaccharide derived from N. meningitidis. In some of these embodiments, the capsular polysaccharide Mn is selected from the group consisting of capsular polysaccharides Mn-serotype A, C, W135 and Y. In certain preferred embodiments, the carrier protein is CRM197 and the capsular polysaccharide is selected from the group consisting of in Mn-serotype A, C, W135 and Y capsular polysaccharides.
[000139] In some embodiments of each of the methods provided here, the saccharide was combined with imidazole or triazole and then reacted with a carbonic acid derivative, such as CDT, in an organic solvent (for example, DMSO) containing about 0.2% w / v water to produce activated saccharides. The use of the combined saccharide in the activation step increases the solubility of the saccharide in the organic solvent. Typically, the saccharide was combined with 10 grams of 1,2,4-triazole excipient per gram of polysaccharide, followed by mixing at room temperature to provide a combined saccharide.
[000140] Thus, in certain embodiments, the methods further comprise a step of combining saccharide with triazole or imidazole to provide a combined saccharide before activation step a). In some of these modalities, the combined saccharide is frozen in a wrap, lyophilized and reconstituted in an organic solvent (such as DMSO) and about 0.2% w / v of water is added before activation with the carbonic acid derivative , for example, CDT.
[000141] In one embodiment, the reaction mixture with the thiolated saccharide is optionally treated with N-acetyl lysine methyl ester for "capping" any unreacted activated saccharide. In some of these modalities, the capping thiolated saccharide mixture is purified by means of ultrafiltration / diafiltration.
[000142] In frequent embodiments, the thiolated saccharide is reacted with a reducing agent to produce an activated thiolated saccharide. In some of these embodiments, the reducing agent is tris (2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT) or mercaptoethanol. In some of these modalities, the activated thiolated saccharide is purified by means of ultrafiltration / diafiltration.
[000143] In one embodiment, the method of producing an eTEC-linked glyco-conjugate comprises the step of adjusting and maintaining the pH of the reaction mixture of activated thiolated saccharide and carrier protein at a pH of about 8 to about 9 for about 20 hours at about 5 ° C.
[000144] In one embodiment, the method of producing a glyco-conjugate of the present invention comprises the step of isolating the thiolated saccharide-carrier protein conjugate after it is produced. In frequent modalities, glycoconjugate is isolated by means of ultrafiltration / diafiltration.
[000145] In another embodiment, the method of producing an eTEC-linked glyco-conjugate the invention comprises the step of isolating the isolated conjugated saccharide carrier protein after being produced. In frequent modalities, glycoconjugate is isolated by means of ultrafiltration / diafiltration.
[000146] In yet another embodiment, the method of producing the activated saccharide comprises the step of adjusting the water concentration of the reaction mixture comprising saccharide and CDT in an organic solvent to between about 0.1 and 0.4%. In one embodiment, the water concentration of the reaction mixture comprising saccharide and CDT in an organic solvent is adjusted to about 0.2%.
[000147] In one embodiment, the saccharide activation step comprises reacting the polysaccharide with an amount of CDT that is a molar excess of about 5 to the amount of polysaccharide present in the reaction mixture comprising capsular polysaccharide and CDT in an organic solvent.
[000148] In another embodiment, the method of producing the glycoconjugate of the invention comprises the step of determining the water concentration of the reaction mixture comprising saccharide. In one such embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in approximately an amount of CDT that is equimolar to the amount of water present in the reaction mixture which comprises saccharide and CDT in an organic solvent.
[000149] In another embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in approximately an amount of CDT that is in a molar ratio of about 0.5: 1 compared to the amount of water present in the reaction mixture comprising saccharide and CDT in an organic solvent. In one embodiment, the amount of CDT added to the reaction mixture to activate the saccharide is provided in approximately an amount of CDT that is in a 0.75: 1 molar ratio compared to the amount of water present in the reaction mixture that comprises saccharide and CDT in an organic solvent.
[000150] In one embodiment, the method comprises the step of isolating the thiolated polysaccharide by means of diafiltration. In another embodiment, the method comprises the step of isolating the activated thiolated polysaccharide by means of diafiltration.
[000151] In one embodiment, the carrier protein used in the production method of a capsular polysaccharide Pn-carrier protein conjugate comprises CRM197. In another embodiment, the carrier protein used in the production method of an isolated Mn-carrier protein capsular polysaccharide comprises CRM197.
[000152] In some embodiments, the saccharide: activated carrier protein (w / w) ratio is between 0.2 and 4. In other embodiments, the saccharide: activated carrier protein (w / w) ratio is between 1.0 and 2 , 5. In other modalities, the ratio of saccharide: activated carrier protein (w / w) is between 0.4 and 1.7. In other modalities, the saccharide: activated carrier protein (w / w) ratio is about 1: 1. In some of these modalities, saccharide is a bacterial capsular polysaccharide and the activated carrier protein is generated by the activation (bromoacetylation) of CRM197.
[000153] In another embodiment, the method of producing the activated saccharide comprises the use of an organic solvent. In frequent embodiments, the organic solvent is a polar aprotic solvent. In some of these modalities, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1 , 3-Dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone (DMPU) and hexamethylphosphoramide (HMPA) or a mixture thereof. In a preferred embodiment, the organic solvent is DMSO.
[000154] In frequent modalities, isolation of the eTEC-linked glycoconjugate comprises an ultrafiltration / diafiltration step.
[000155] In one embodiment, the saccharide used in the glycoconjugate production method of the present invention has a molecular weight between about 10 kDa and about 2,000 kDa. In another embodiment, the saccharide used in the glycoconjugate production method of the present invention has a molecular weight between about 50 kDa and about 2,000 kDa.
[000156] In one embodiment, the glycoconjugate produced in the glycoconjugate production method of capsular polysaccharide-carrier protein has a size between about 50 kDa and about 20,000 kDa. In another embodiment, the glycoconjugate produced in the glycoconjugate production method of capsular polysaccharide-carrier protein has a size between about 500 kDa and about 10,000 kDa. In one embodiment, the glycoconjugate produced in the glycoconjugate production method of capsular polysaccharide-carrier protein has a size between about 1,000 kDa and about 3,000 kDa.
[000157] In another aspect, the invention provides a glycoconjugate comprising an eTEC-linked saccharide conjugated to a carrier protein by means of an eTEC spacer produced by any of the methods described here.
[000158] In another aspect, the invention provides an immunogenic composition comprising an eTEC-linked glycoconjugate produced by any of the methods described herein.
[000159] The degree of saccharide O-acetylation can be determined by any method known in the art, for example, through proton NMR (Lemercinier and Jones (1996) Carbohydrate Research 296; 83-96, Jones and Lemercinier ( 2002) J. Pharmaceutical and Biomedical Analysis 30; 1233-1247, WO 05/033148 or WO 00/56357). Another commonly used method is described by Hestrin (1949) J. Biol. Chem. 180; 249-261. Yet another method is based on ion exclusion chromatography-HPLC. The degree of O-acetylation is determined by evaluating the amount of free acetate present in a sample and comparing this value with the amount of acetate released after a hydrolysis with a soft base. Acetate is decomposed from other components of the sample and quantified with ultraviolet (UV) detection at 210 nm. Another method is based on ion exclusion chromatography - HPLC. O-acetyl is determined by evaluating the amount of free acetate present in a sample and comparing this value with the amount of acetate released after a hydrolysis with a soft base. Acetate is decomposed from other components of the sample and quantified with ultraviolet (UV) detection at 210 nm. Degree of Conjugation Determined Through Amino Acid Analysis
[000160] Acid hydrolysis of "IAA pre-capping" conjugate samples generated using bromoacetyl activation chemistry resulted in the formation of acid-stable S-carboxymethylcysteine (CMCA) from the cystamine at the conjugated and S-carboxymethylcysteine (CMC) sites acid-stable from cysteines at sites that have been capped. Acid hydrolysis of the "post-capping IAA" conjugates (finals) generated using bromoacetyl activation chemistry resulted in the formation of acid-stable S-carboxymethylcysteamine (CMCA) from cystamine at the IAA capping and conjugated sites and acid-stable S-carboxymethylcysteine (CMC) from cysteines at capped sites. All non-conjugated and uncapped lysines were again converted to lysine and detected as such. All other amino acids were again hydrolyzed to free amino acids, except for tryptophan and cysteine, which were destroyed by hydrolysis conditions. Asparagine and glutamine were converted to aspartic acid and glutamic acid, respectively.
[000161] The amino acids of each hydrolyzed and control sample were separated using ion exchange chromatography, followed by reaction with a Beckman Ninhydrin NinRX solution at 135 ° C. The derivatized amino acids were then detected in the visible range at 570 nm and 440 nm (see Table 1). A standard set of amino acids [Pierce Amino Acid Standard H] containing 500 picomoles of each amino acid was passed along with the samples and controls for each set of analysis. S-carboxymethylcysteine [Sigma-Aldrich] was added to the standard. Table 1
[000162] Retention Times for Amino Acids Using Gradient Program 1 on Beckman 6300 Amino Acid Analyzer


[000163] Lysine was chosen for evaluation based on its covalent bond to cysteine and cysteamine and the expected similar hydrolysis. The resulting mole numbers of amino acids were compared with the amino acid composition of the protein and recorded, along with the values for CMC and CMCA. The CMCA value was used directly to assess the degree of conjugation and the CMC value was used directly to assess the degree of "capping".
[000164] In one embodiment, the glycoconjugate is characterized by its molecular size distribution (Kd). The molecular size of the conjugates is determined by size exclusion chromatography (SEC - Size Exclusion Chromatography) in a stationary phase in Sepharose CL-4B medium using a high pressure liquid chromatography system (High Pressure Liquid Chromatography - HPLC). For Kd determination, the chromatography column is first calibrated to determine Vo, which represents the void volume or total exclusion volume, and V, the volume at which the smallest molecules in the sample elute, which is also known as the interparticle volume. Every SEC separation occurs between Vo and V. The Kd value for each fraction collected is determined by the following expression: Kd = (Ve-Vi) Z (V-Vo), where Ve represents the retention volume of the compound. The fraction% (main peak) that elutes 0.3 defines the Kd (molecular size distribution) of the conjugate. Immunogenic Compositions
[000165] The term "immunogenic composition" refers to any pharmaceutical composition containing an antigen (for example, a microorganism or a component thereof) that can be used to induce an immune response in an individual.
[000166] As used here, "immunogenic" means the ability of an antigen (or an epitope of the antigen), such as a bacterial capsular polysaccharide or a glycoconjugate or immunogenic composition comprising a bacterial capsular polysaccharide, to induce an immune response in a host individual, such as a mammal, either humorally or cellularly mediated or both.
[000167] Glycoconjugate can serve to sensitize the host by presenting the antigen in association with MHC molecules on the surface of a cell. In addition, antigen-specific T cells or antibodies can be generated to allow future protection of an immunized host. Glycoconjugates can thus protect the host against one or more symptoms associated with infection by bacteria or 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 the death of bacteria in an animal efficacy model or through an opsonophagocytic death assay. Methods for Inducing an Immune Response and Protection Against Infection
[000168] The present invention also includes methods for using eTEC-linked glycoconjugates and immunogenic compositions comprising them, whether prophylactically or therapeutically. For example, an aspect of the invention provides a method of inducing an immune response against pathogenic bacteria, for example, pneumococcal or meningococcal bacteria, comprising administering to an individual an immunologically effective amount of any of the immunogenic compositions described herein comprising a bacterial antigen, such as a bacterial capsular polysaccharide derived from pathogenic bacteria. An embodiment of the invention provides a method to protect an individual against infection by pathogenic bacteria or a method of preventing, treating or ameliorating a disease or condition associated with infection by pathogenic bacteria or a method of reducing the severity or delaying the onset of hair at least one symptom associated with an infection caused by pathogenic bacteria, in each case methods comprising administering to an individual an immunologically effective amount of any of the immunogenic compositions described herein comprising a bacterial antigen, such as a bacterial capsular polysaccharide derived of pathogenic bacteria.
[000169] An embodiment of the invention provides a method of preventing, treating or ameliorating an infection, disease or bacterial condition in an individual comprising administering to the individual an immunologically effective amount of an immunogenic composition of the invention, wherein said composition immunogenic comprises an eTEC-linked glycoconjugate comprising a bacterial antigen, such as a bacterial capsular polysaccharide.
[000170] In some modalities, the method of preventing, treating or ameliorating an infection, disease or bacterial condition comprises human, veterinary, animal or agricultural treatment. Another modality provides a method of preventing, treating or ameliorating an infection, disease or bacterial condition associated with pathogenic bacteria in an individual, the method comprising generating a polyclonal antibody or monoclonal antibody prepared from the immunogenic composition described here and using said preparation of antibodies to confer passive immunity to the individual. One embodiment of the invention provides a method of preventing a bacterial infection in an individual undergoing a surgical procedure, the method comprising the step of administering a prophylactically effective amount of an immunogenic composition described herein to the individual prior to the surgical procedure.
[000171] In preferred embodiments of each of the preceding processes, the pathogenic bacteria are pneumococcal or meningococcal bacteria, such as S. pneumoniae or N. meningitidis bacteria. In some of these modalities, the bacterial antigen is a capsular polysaccharide selected from the group consisting of Pn capsular polysaccharides of serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14 , 15B, 18C, 19A, 19F, 22F, 23F and 33F. In other embodiments, the bacterial antigen is a capsular polysaccharide selected from the group consisting of capsular polysaccharides Mn of serotype A, C, W135e Y.
[000172] An immune response to an antigen or immunogenic composition is characterized by the development, in an individual, of a humoral response and / or a cell-mediated immune response to the molecules present in the immunogenic composition or antigen of interest. For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and involves the induction and production of antibodies that recognize and bind with some affinity to the antigen in the immunogenic composition of the invention, while an "immune-mediated immune response" per cell "is mediated by T cells and / or other white blood cells. An "immune response mediated by cells" is caused by the presentation of antigenic epitopes in association with molecules of the major histocompatibility complex (Major Histocompatibility - MHC) of Class I or Class II, CD1 or other non-classical MHC type molecules. This activates antigen-specific helper CD4 + T cells or CD8 + cytotoxic T lymphocyte cells ("CTLs"). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by classical or non-classical MHCs and expressed on cell surfaces. CTLs help to induce and promote the intracellular destruction of intracellular microorganisms or the lysis of cells infected by such microorganisms. Another aspect of cellular immunity involves a specific antigen response by helper T cells. Helper T cells act by helping to stimulate the function and concentrate the activity of non-specific effector cells against cells that exhibit peptide or other antigens in association with classic 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 + and 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 by lymphoproliferation assays (lymphocyte activation), CTL cytotoxic cell assays, lymphocyte assays Specific antigen T in a sensitized individual or by measuring cytokine production by T cells in response to new stimulation with the antigen. Such assays are well known in the art. See, for example, Erickson etal. (1993) J. Immunol. 151: 4189-4199; and Doe etal. (1994) Eur. J. Immunol. 24: 2369-2376.
[000173] The immunogenic compositions and methods of the invention may be useful for one or more of the following procedures: (i) preventing infection or reinfection, such as in a traditional vaccine, (ii) reducing the severity of or eliminating symptoms and / or (iii) substantial or complete elimination of the pathogen or disorder in question. Consequently, treatment can be carried out prophylactically (before infection) or therapeutically (after infection). In the present invention, prophylactic treatment is the preferred mode. In accordance with a particular embodiment of the present invention, compositions and methods are provided which treat, including prophylactically and / or therapeutically immunizing an individual host against bacterial infection, for example, by S. pneumoniae or N. meningitidis. The methods of the present invention are useful for imparting prophylactic and / or therapeutic immunity to an individual. The methods of the present invention can also be practiced on individuals for bio-medical research applications.
[000174] As used here, the term "individual" means a human or non-human animal. More particularly, an individual refers to any animal classified as a mammal, including humans, domestic and farmed animals, research, zoo, sport and companion animals, pets, such as a domestic pet and others domesticated animals including, but not limited to, cattle, sheep, ferrets, pigs, horses, rabbits, goats, dogs, cats and the like. Favorite pets are dogs and cats. Preferably, the individual is a human being.
[000175] The amount of a particular conjugate in a composition is generally calculated based on the total amount of polysaccharide, both conjugated and unconjugated for this conjugate. For example, a conjugate with 20% free polysaccharides will have about 80 pg of polysaccharide conjugate and about 20 pg of unconjugated polysaccharide in a 100 pg polysaccharide dose. The contribution of the protein to the conjugate is not usually considered when calculating the doses of a conjugate. The immunogenic amount of an immunogenic composition or conjugate can vary depending on the bacterial serotype. Generally, each dose comprises 0.1 to 100 pg of the polysaccharide, particularly 0.1 to 10 mg and, more particularly, 1 to 10 pg. The immunogenic amount of the different polysaccharide components in an immunogenic composition may differ and each may comprise 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 15 pg , 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg or about 100 pg of any particular polysaccharide antigen.
[000176] The term "invasive disease" refers to the isolation of bacteria from a normally sterile location, where there are associated clinical symptoms / signs of disease. Normally, sterile body parts include blood, CSF, pleural fluid, pericardial fluid, peritoneal fluid, joint / synovial fluid, bone, internal parts of the body (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 so on.
[000177] The effectiveness of an antigen as an immunogen can be measured by proliferation assays, by cytolytic assays, such as chromium release assays to measure the ability of a T cell to subject the specific target cell to lysis, or by assessing levels of B cell activity by measuring the levels of circulating antibodies in the serum specific for the antigen. An immune response can also be detected by measuring the serum levels of specific antigen antibodies induced after administration of the antigen and, more specifically, by measuring the ability of the antibodies thus induced to increase the opsonophagocytic capacity of specific white blood cells, as described here. 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 for 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 the percentage of survival or the percentage of mortality after challenge of the animals with the cells bacterial. In one modality, the amount of protection can be assessed 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 vaccine with multiple antigens or vaccine with multiple components or immunogenic compositions will vary in relation to each of the other components and can be determined by methods known to those skilled in the art. Such methods include procedures for measuring immunogenicity and / or effectiveness in vivo.
[000178] In another aspect, the invention provides antibodies and antibody compositions that bind specifically and selectively to the capsular or glycoconjugate polysaccharides of the present invention. In some of such embodiments, the invention provides antibodies and antibody compositions that bind specifically and selectively to Pn capsular polysaccharides of serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F or 33F or glycoconjugates comprising the same. In other embodiments, the invention provides antibodies and antibody compositions that specifically and selectively bind to capsular polysaccharides Mn of serotype A, C, W135 or Y or glycoconjugates comprising them. In some embodiments, antibodies are generated by administering, to an individual, the capsular or glycoconjugate polysaccharides of the present invention. In some embodiments, the invention provides purified or isolated antibodies directed against one or more of the capsular or glycoconjugate polysaccharides of the present invention. In some embodiments, the antibodies of the present invention are functional, as measured by killing bacteria in any animal efficacy model or through an opsonophagocytic death assay. The antibodies or antibody compositions of the invention can be used in a method of treating or preventing an infection, disease or bacterial condition associated with pathogenic bacteria in an individual, for example, S. pneumoniae or N. meningitidis bacteria, the method comprising generation of a polyclonal antibody or monoclonal antibody preparation and use of said antibody or antibody composition to impart passive immunity to the individual. The antibodies of the invention can also be useful for diagnostic methods, for example, detecting the presence or quantification of the levels of capsular polysaccharide or a glycoconjugate thereof. For example, the antibodies of the invention can also be useful for detecting the presence or quantification of the levels of a Pn or Mn capsular polysaccharide or even a glycoconjugate, wherein the glycoconjugate comprises the bacterial capsular polysaccharide conjugated to a carrier protein using a spacer eTEC.
[000179] Various assays and animal models known in the art can be used to evaluate the effectiveness of any of the immunogenic compositions described here. For example, Chiavolini et al. Clin. Microbiol. Rev. (2008), 21 (4): 666-685) describe animal models of S. pneumoniae diseases. Gorringe et al. METHODS IN MOLECULAR MEDICINE, vol. 66 (2001), Chapter 17, Pollard and Maiden eds. (Humana Press Inc.) describe animal models for meningococcal diseases. Opsonophagocytic Activity Assay (OPA)
[000180] OPA test procedures were based on the methods previously described by Hu, et al. (Clin. Diaqn. Lab. Immunol. 2005; 12 (2): 287-95), with the following modifications. Thermally inactivated sera were serially diluted 2.5 times in buffer. Target bacteria were added to the assay plates and incubated for 30 min at 25 ° C on a shaker. Baby rabbit complement (3 to 4 weeks of age, Pel-Freez, 12.5% final concentration) and differentiated HL-60 cells were then added to each well at a ratio of approximately 200: 1 effector to target . The assay plates were incubated for 45 minutes at 37 ° C on a shaker. To finish the reaction, 80 pL of 0.9% NaCI was added to all wells, mixed and a 10 pL aliquot was transferred to wells of plates with Millipore MultiScreenHTS HV filter containing 200 pL of water. Liquid was filtered through the plates under vacuum and 150 µl of HySoy medium was added to each well and filtered. The filter plates were then incubated at 37 ° C, 5% CO2 overnight and were then fixed with Destain's solution (Bio-Rad). The plates were then stained with Coomasie blue and bleached once. Colonies were photographed and listed in an Immunospot Analyzer® from Cellular Technology Limited (CTL). The titration of OPA antibodies was interpolated from the reciprocal of the two serum dilutions covering the point of 50% reduction in the number of bacterial colonies compared to the control wells that did not contain immune serum.
[000181] The foregoing description generically describes the present invention. A more complete understanding can be obtained by reference to the specific examples below. These examples are described for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLES
[000182] Example 1. General Process for Preparation of Glycoconjugates Linked by eTEC Activation of Saccharide and Thiolation with Cystamine Dihydrochloride
[000183] The saccharide is reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the solution is determined by Karl Fischer analysis (KF) and adjusted to achieve a moisture content of 0.1 and 0.4%, typically 0.2%.
[000184] To initiate activation, a solution of 1,1-carbonyl-di-1,2,4-triazole (CDT) or 1, T-carbonyldiimidazole (CDI) is prepared fresh in a concentration of 100 mg / mL in DMSO. The saccharide is activated with various amounts of CDT / CDI (1-10 molar equivalents) and the reaction is allowed to proceed for 1 hour at 23 ± 2 ° C. The level of activation can be determined by HPLC. Cystamine dihydrochloride is prepared in anhydrous DMSO at a concentration of 50 mg / mL. The activated saccharide is reacted with 1 eq. mol. of cystamine dihydrochloride. Alternatively, the activated saccharide is reacted with 1 eq. mol of cysteamine hydrochloride. The thiolation reaction is allowed to proceed for 21 ± 2 hours at 23 ± 2 ° C to produce a thiolated saccharide. The level of thiolation is determined by the added amount of CDT / CDI.
[000185] Residual CDT / CDI in the activation reaction solution are dissipated by adding 100 mM sodium tetraborate solution, pH 9.0. Calculations are performed to determine the amount of tetraborate added and adjust the final moisture content to be up to 1-2% of the total aqueous solution. Reduction and Purification of Activated Thiol Saccharide
[000186] The thiolated saccharide reaction mixture is diluted 10 times by adding 5 mM sodium succinate in pre-cooled 0.9% saline, pH 6.0, and filtered through a 5 pm filter . Dialfiltration of the thiolated saccharide is performed against a 40-fold WFI diavolume. Upon retentation, a solution of tris (2-carboxyethyl) phosphine (TCEP), 1-5 eq. mol., is added after dilution in a 10% volume of 0.1 M sodium phosphate buffer, pH 6.0. This reduction reaction is allowed to proceed for 20 ± 2 hours at 5 ± 3 ° C. Purification of the activated thiolated saccharide is preferably carried out by means of ultrafiltration / dialfiltration against pre-cooled 10 mM monobasic sodium phosphate, pH 4.3. Alternatively, the thiolated saccharide is purified by conventional size exclusion chromatography (SEC) procedures or ion exchange chromatographic methods. An aliquot of activated thiolate saccharide retentate is extracted to determine the saccharide concentration and thiol content assays (Ellman). Alternative Reduction and Purification of Activated Thiol Saccharide
[000187] As an alternative to the purification procedure described above, activated thiolated saccharide was also purified as follows.
[000188] To the reaction mixture of thiolated saccharide, a solution of tris (2-carboxyethyl) phosphine (TCEP), 5 - 10 eq. mol., is added and allowed to proceed for 3 ± 1 hours at 23 ± 2 ° C. The reaction mixture was then diluted 5 times by adding 5 mM sodium succinate in pre-cooled 0.9% saline, pH 6.0, and filtered through a 5 pm filter. Dialfiltration of thiolated saccharide was performed using a 40-fold diavolume of pre-cooled 10 mM sodium phosphate, pH 4.3. An aliquot of activated thiolate saccharide retentate was extracted to determine the saccharide concentration and thiol content assays (Ellman). Activation and Purification of Bromoacetylated Carrier Protein
[000189] Free amino groups of the carrier protein are bromoacetylated by reaction with a bromoacetylating agent, such as bromoacetic acid N-hydroxysuccinimide ester (BAANS), bromoacetyl bromide or other suitable reagent.
[000190] The carrier protein (in 0 to 1 M sodium phosphate, pH 8.0 ± 0.2) is first maintained at 8 ± 3 ° C at a pH of about 7 before activation. To the protein solution, the N-hydroxysuccinimide ester of bromoacetic acid (BAANS) as a stock solution in dimethyl sulfoxide (DMSO) (20 mg / mL) is added in a BAANS: protein ratio of 0.25: 0, 5 (w / w). The reaction is mixed gently at 5 + 3 ° C for 30 - 60 minutes. The resulting bromoacetylated (activated) protein is purified, for example, by ultrafiltration / diafiltration using a 10 kDa MWCO membrane using 10 mM phosphate buffer (pH 7.0). After purification, the protein concentration of the bromoacetylated carrier protein is estimated by the Lowry protein assay.
[000191] The degree of activation is determined by the total bromide assay using ion exchange liquid chromatography coupled with suppressed conductivity detection (ion chromatography). The bromide bound on the activated bromoacetylated protein is cleaved from the protein when preparing test samples and quantified together with any free bromide that may be present. Any remaining covalently bound bromine in the protein is released by converting ionic bromide by heating the sample to alkaline 2-mercaptoethanol. CRM197 Bromoacetylated Activation and Purification
[000192] CRM197 was diluted to 5 mg / mL with 10 mM phosphate buffered with 0.9% NaCI, pH 7 (PBS) and then 0.1 M NaHCOs, pH 7.0, using 1 M. stock solution BAANS was added in a CRM: BAANS ratio of 1: 0.35 (w / w) using a 20 mg / mL BAANS stock solution in DMSO. The reaction mixture was incubated at 3 ° C to 11 ° C for 30 minutes-1 hour, then purified by ultrafiltration / diafiltration using a 10K MWCO membrane and 10 mM sodium phosphate / 0 NaCI , 9%, pH 7.0. The purified activated CRM197 was tested by the Lowry assay to determine the protein concentration and then diluted with PBS to 5 mg / mL. Sucrose was added at 5% w / v as a cryoprotectant and the activated protein was frozen and stored at 25 ° C until needed for conjugation.
[000193] Bromoacetylation of CRM197 lysine residues was very consistent, resulting in the activation of 15 to 25 lysines out of the 39 available lysines. The reaction produced high yields of activated protein. Conjugation of Thiolated Saccharide Activated to Bromoacetylated Carrier Protein
[000194] Before starting the conjugation reaction, the reaction vessels are pre-cooled to 5 ° C. Bromoacetylated carrier protein and activated thiolated saccharide are subsequently added and mixed at a stirring speed of 150-200 rpm. The saccharide / protein intake ratio is 0.9 ± 0.1. The reaction pH is adjusted to 8.0 ± 0.1 with 1 M NaOH solution. The conjugation reaction is allowed to proceed at 5 ° C for 20 ± 2 hours. Capping of Residual Reactive Functional Groups
[000195] The unreacted bromoacetylated residues in the carrier protein are dissipated by means of a reaction with 2 eq. mol. of N-acetyl-L-cysteine as a capping reagent for 3 hours at 5 ° C. Residual free sulfhydryl groups are capped at 4 eq. mol of iodoacetamide (IAA) for 20 hours at 5 ° C. Purification of eTEC-Linked Glycoconjugate
[000196] The conjugation reaction mixture (post-capping with IAA) is filtered through a 0.45 pm filter. Ultrafiltration / dialfiltration of glycoconjugate is performed against 5 mM succinate-0.9% saline, pH 6.0. The glycoconjugate retentate is then filtered through a 0.2 pm filter. An aliquot of glycoconjugate is extracted for testing. The remaining glycoconjugate is stored at 5 ° C.
[000197] Example 2. Preparation of Pn-33F eTEC Conjugates Activation Process Pn-33F Polysaccharide Activation
[000198] Polysaccharide Pn-33F was combined with 1,2,4-triazole at 500 mM (in WFI) to obtain 10 grams of triazole per gram of polysaccharide. The mixture was frozen in a wrapper in a dry ice-ethanol bath and then lyophilized until dry. The lyophilized 33F polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized 33F / DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the 33F / DMSO solution to achieve a moisture content of 0.2%.
[000199] To initiate activation, 1,1 '-carbonyl-di-1,2,4-triazole (CDT) was prepared fresh as 100 mg / mL in DMSO solution. Polysaccharide Pn-33F was activated with various amounts of CDT before the thiolation step. Activation with CDT was performed at 23 ± 2 ° C for 1 hour. The activation level was determined by HPLC (A220 / A205). 100 mM sodium tetraborate solution, pH 9.0, was added to dissipate any residual CDT in the activation reaction solution. Calculations are performed to determine the amount of tetraborate added and allow the final moisture content to be 1.2% of total aqueous solution. The reaction was allowed to proceed for 1 hour at 23 ± 2 ° C. Activated Pn-33F Polysaccharide Thiol
[000200] Cystamine dihydrochloride was prepared fresh in anhydrous DMSO and 1 eq. mol. of cystamine dihydrochloride was added to the activated polysaccharide reaction solution. The reaction was allowed to proceed for 21 ± 2 hours at 23 ± 2 ° C. The thiolated saccharide solution was diluted 10 times by adding 5 mM sodium succinate in pre-cooled 0.9% saline, pH 6.0. The diluted reaction solution was filtered through a 5 pm filter. Thiolated Pn-33F polysaccharide dialfiltration was performed with 100K MWCO ultrafiltration membrane cassettes using water for injection (Water For Injection - WFI).
[000201] The level of thiolation of activated Pn-33F polysaccharides as a function of molar equivalents of CDT is shown in Figure 8. Reduction and Purification of Activated Thiolate Pn-33F Polysaccharide
[000202] Upon retentation, a solution of tris (2-carboxyethyl) phosphine (TCEP), 5 eq. mol., was added after dilution in a 10% volume of 0.1 M sodium phosphate buffer, pH 6.0. This reduction reaction was allowed to proceed for 2 hours at ± 1 23 ± 2 ° C. Dialfiltration of thiolated 33F polysaccharide was performed with 100K MWCO ultrafiltration membrane cassettes. Diafiltration was performed against pre-cooled 10 mM sodium phosphate, pH 4.3. The thiolate 33F polysaccharide retentate was extracted for assays of saccharide and thiol concentration (Ellman). Alternative Reduction and Purification of Activated Thiolate Pn-33F Polysaccharide
[000203] As an alternative to the purification procedure described above, thiolated activated 33F saccharide was also purified as follows.
[000204] To the reaction mixture of thiolated saccharide, a solution of tris (2-carboxyethyl) phosphine (TCEP), 5 eq. mol., was added and allowed to proceed for 3 ± 1 hours at 23 ± 2 ° C. The reaction mixture was then diluted 5 times by adding 5 mM sodium succinate in pre-cooled 0.9% saline, pH 6.0, and filtered through a 5 pm filter. Dialfiltration of thiolated saccharide was performed using a 40-fold diavolume of pre-cooled 10 mM sodium phosphate, pH 4.3, with 100K MWCO ultrafiltration membrane cassettes. The thiolate 33F polysaccharide retentate was extracted for assays of saccharide and thiol concentration (Ellman). A flowchart of the activation process is provided in Figure 7 (A). Conjugation Process Conjugation of Pn-33F Thiolated polysaccharide to CRM197 Bromoacetylated
[000205] The CRM197 carrier protein was activated separately by means of bromoacetylation as described in Example 1 and then reacted with the activated Pn-33F polysaccharide for the conjugation reaction. Before starting the conjugation reaction, the reactor was pre-cooled to 5 ° C. CRM197 bromoacetylated and thiolated 33F polysaccharide were mixed together in a reaction vessel at a stirring speed of 150-200 rpm. The proportion of saccharide / protein intake was 0.9 ± 0.1. The reaction pH was adjusted to 8.0-9.0. The conjugation reaction was allowed to proceed at 5 ° C for 20 ± 2 hours. Capping of Reactive Groups in CRM197 Bromoacetylated and Pn-33F Thiolate polysaccharide
[000206] The unreacted bromoacetylated residues on CRM197 proteins were "capped" by means of a reaction with 2 eq. mol. of N-acetyl-L-cysteine for 3 hours at 5 ° C, followed by "capping" of any residual free sulfhydryl groups of the thiolated polysaccharide 33F with 4 eq. mol. iodoacetamide (IAA) for 20 hours at 5 ° C. Purification of Pn-33F Glycoconjugate Connected by eTEC
[000207] The conjugation solution was filtered through a 0.45 pm or 5 pm filter. Dialfiltration of the 33F glycoconjugate was performed with 300K MWCO ultrafiltration membrane cassettes. Disfiltration was performed against 5 mM succinate-0.9% saline, pH 6.0. The 300K retentate of the Pn-33F glycoconjugate was then filtered through a 0.22 pm filter and stored at 5 ° C.
[000208] A flow chart of the conjugation process is provided in Figure 7 (B). Results
[000209] The reaction parameters and characterization data for several batches of Pn-33F eTEC glycoconjugates are shown in Table 2. The activation-thiolation of CDT with cystamine dihydrochloride generated glycoconjugates having saccharide yields of 63 to 90% and < 1% to 13% free saccharides. Table 2. Experimental Parameters and Characterization Data for Pn-33F eTEC Conjugates

OPA titrations in Pn-33F eTEC glycoconjugates to CRM197
[000210] OPA titrations in Pn-33F in mice were determined under conventional conditions. OPA titrations (GMT with 95% CI) at four and seven weeks are shown in Table 3, demonstrating that the Pn-33F serotype glycoconjugate induced OPA titrations in a murine immunogenicity model. Table 3. OPA titrations in PN-33F (GMT with 95% CI)
Example 3. Preparation of Pn-22F eTEC Conjugates Activation Process Pn-22F Polysaccharide Activation
[000211] Polysaccharide Pn-22F was combined with 1,2,4-triazole at 500 mM (in WFI) to obtain 10 grams of triazole per gram of polysaccharide. The mixture was frozen in a wrapper in a dry ice-ethanol bath and then lyophilized until dry. The lyophilized 22F polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). The moisture content of the lyophilized 22F / DMSO solution was determined by Karl Fischer (KF) analysis. The moisture content was adjusted by adding WFI to the Pn-22F / DMSO solution to achieve a moisture content of 0.2%.
[000212] To initiate activation, 1, T-carbonyl-di-1,2,4-triazole (CDT) was prepared fresh as 100 mg / mL in DMSO solution. Pn-22F polysaccharide was activated with various amounts of CDT, followed by thiolation with 1 eq. mol. of cystamine dihydrochloride. Activation with CDT was performed at 23 ± 2 ° C for 1 hour. The activation level was determined by HPLC (A220 / A205). Sodium tetraborate solution, 100 mM, pH 9.0, was added to dissipate any residual CDT in the activation reaction solution. Calculations are performed to determine the amount of tetraborate added and allow the final moisture content to be 1.2% of total aqueous solution. The reaction was allowed to proceed for 1 hour at 23 ± 2 ° C. Activated Pn-22F Polysaccharide Thiolation
[000213] Cystamine dihydrochloride was prepared fresh in anhydrous DMSO and added to the reaction solution. The reaction was allowed to proceed for 21 ± 2 hours at 23 ± 2 ° C. The thiolated saccharide solution was diluted 10 times by adding 5 mM sodium succinate in pre-cooled 0.9% saline, pH 6.0. The diluted reaction solution was filtered through a 5 pm filter. Thiolate Pn-22F polysaccharide dialfiltration was performed with 100K MWCO ultrafiltration membrane cassettes using water for injection (Water For Injection - WFI). Reduction and Purification of Pn-22F Activated Thiolated Polysaccharide
[000214] Upon retentation, a solution of tris (2-carboxyethyl) phosphine (TCEP), 5 - 10 eq. mol., was added after dilution in a 10% volume of 0.1 M sodium phosphate buffer, pH 6.0. This reduction reaction was allowed to proceed for 2 ± 1 hour at 23 ± 2 ° C. Diafiltration of thiolated 22F polysaccharide was performed with 100K MWCO ultrafiltration membrane cassettes. Diafiltration was performed against pre-cooled 10 mM sodium phosphate, pH 4.3. The thiolate 22F polysaccharide retentate was extracted for assays of saccharide and thiol concentration (Ellman). PN-22F eTEC Glycoconjugate Conjugation, Capping and Purification
[000215] Conjugation of the activated thiolated Pn22F polysaccharide to the activated CRMi97, "capping" and purification of the Pn-22F eTEC glycoconjugates were performed according to the procedures described in Example 2. Results
[000216] Characterization data and representative processes for glycoconjugates from Pn 22F-ETEC to CRM197 are provided in Table 4. Table 4. Experimental Parameters and Characterization Data for Pn-22F eTEC Conjugates

Example 4. Preparation of Pn-10A eTEC Conjugates to CRM197 Preparation of PN-10A eTEC Glycoconjugates
[000217] Glycoconjugates comprising the serotype 10A pneumococcal capsular polysaccharide (10A-Pn) conjugated to CRM197 by means of the eTEC spacer were prepared according to the procedures described in Example 2. Characterization of PN-10A eTEC glycoconjugates
[000218] Characterization data and representative processes for glycoconjugates from Pn-10A eTEC to CRM197 are provided in Table 5. Table 5. Experimental parameters and characterization data for PN-10ª glycoconjugates

OPA titrations in Pn-10A
[000219] OPA titrations against the Pn-10A eTEC conjugate to CRM197 in mice were determined under conventional conditions. OPA titrations as a function of dose are shown in Table 6. OPA titrations were significantly higher for the conjugate compared to the unconjugated serotype 10A polysaccharide. Table 6. OPA titrations in PN-10A (GMT with 95% CI)
Example 5. Preparation of Pn-11A eTEC Conjugates to CRM197 Preparation of PN-11A eTEC Glycoconjugates
[000220] Glycoconjugates comprising the serotype 11A pneumococcal capsular polysaccharide (11 A-Pn) conjugated to CRM197 using the eTEC spacer were prepared according to the procedures described in Example 2. Characterization of PN-11A eTEC glycoconjugates
[000221] Characterization data and representative processes for glycoconjugates from Pn-11A ETEC to CRM197 are provided in Table 7. Table 7. Experimental parameters and characterization data for glycoconjugates from PN-11ª
OPA titrations in Pn-11A
[000222] OPA titrations against Pn-11A eTEC conjugate to CRM197 in mice were determined under conventional conditions. OPA titrations as a function of dose are shown in Table 8. Table 8. OPA titrations in Pn-11A (GMT with 95% CI)
Example 6. Preparation of Pn-33F RAC / Aqueous conjugates to CRM197 Preparation of Pn-33F RAC / Aqueous Glycoconjugates
[000223] Pn-33F glycoconjugates were prepared using reductive aqueous phase (RAC / aqueous) amination, which has been successfully applied to produce a pneumococcal conjugate vaccine (see, for example, WO 2006/110381) . This approach comprises two steps. The first step is polysaccharide oxidation to generate aldehyde functionality from vicinal diols. The second step is to conjugate the activated polysaccharide to the lysine residues (Lys) of CRM 197-
[000224] Briefly, frozen polysaccharide was thawed and oxidation was carried out in sodium phosphate buffer at a pH of 6.0 by adding different amounts of sodium periodate (NalOzi). Concentration and diafiltration of the activated polysaccharide were performed and the purified activated polysaccharide was stored at 4 ° C. Activated polysaccharide was combined with the CRM197 protein. Complete mixing of the polysaccharide and CRM197 is performed before placing the vial in a dry ice / ethanol bath, followed by lyophilization of the polysaccharide / CRMi97 mixture. The lyophilized mixture was reconstituted in 0.1 M sodium phosphate buffer. Conjugation reaction was initiated by adding 1.5 molar equivalents of sodium cyanoborohydride and incubating for 20 hours at 23 ° C and another 44 hours at 37 ° C. The reactions were diluted with a volume of 1x 0.9% saline and capping performed with 2 mEq of sodium borohydride for 3 hours at 23 ° C. The reaction mixture was diluted with a volume of 1x 0.9% saline and then filtered through a 0.45 pm filter before purification. Concentration and diafiltration of the conjugate were performed using UF membrane cassettes with 100K MWCO.
[000225] Various conjugates were obtained using the process described above by varying different parameters (for example, pH, reaction temperature and polysaccharide concentration).
[000226] The typical polysaccharide yield was approximately 50% for these conjugates and 15% free saccharides with one MW of the conjugate in the 2000-3500 kDa range.
[000227] However, the native 33F serotype polysaccharide contains a C2 O-acetyl group from the 5-galactofuranose residue and ~ 80% of the acetyl functional groups have been found to be removed during the conjugation process using amination reductive in aqueous phase. It was observed that the O-acetyl group in the five-element ring structure (5-galactofuranoside) can easily migrate and be removed using reductive amination chemistry in the aqueous phase process. Pn-33F RAC / Aqueous Glycoconjugate Stability Assessment
[000228] Aliquots of representative RAC / aqueous conjugates prepared by the process described above were placed in polypropylene tubes. These tubes were stored at 25 ° C or 37 ° C and stability was monitored for up to 3.5 months. At each stability time point,% free saccharide levels were assessed. The stability data at both temperatures are summarized in Table 9. As shown in Table 9, the% free saccharide levels increased significantly at 25 ° C and 37 ° C. Increase in free saccharide% levels during storage is a potential indicator of polysaccharide degradation in the conjugate. Table 9: Stability data for RAC / Aqueous conjugate at 25 ° C and 37 ° C

wk = week; M = month.
[000229] Although the serotype 33F polysaccharide has been successfully activated by reaction with sodium periodate and subsequently conjugated to CRM197 exploring the aqueous reductive amination chemistry, the results of% free saccharide stability under conditions accelerated combined with the inability to preserve acetyl functionality (a key polysaccharide epitope for immunogenicity) during conjugation suggested that the RAC / aqueous process is not the ideal process for conjugating the serotype 33F. Example 7. Preparation of Pn-33F RAC / DMSO Conjugates to CRM197 Preparation of Pn-33F RAC / DMSO Glycoconjugates
[000230] Compared to the RAC / aqueous process, conjugation performed via reductive amination in DMSO (RAC / DMSO) generally has a significantly less chance of de-O-acetylation. In view of the challenges associated with preserving O-acetyl functionality using the RAC / aqueous process described in Example 6, an alternative approach using RAC / DMSO solvent, which has been successfully applied to produce a pneumococcal conjugate vaccine ( see, for example, WO 2006/110381) has been evaluated.
[000231] Activated polysaccharide was combined with sucrose (50% w / v in WFI) using a ratio of 25 grams of sucrose per gram of activated polysaccharide. The components were mixed well before freezing in a wrap in a dry ice / ethanol bath. The flask with frozen mixed mixture wrap was then lyophilized until dry.
[000232] Lyophilized activated polysaccharide was reconstituted in dimethyl sulfoxide (DMSO). DMSO was added to lyophilized CRM197 for reconstitution. Reconstituted activated polysaccharide was combined with reconstituted CRM197 in the reaction vessel. Conjugation was initiated by adding NaCNBHs to the reaction mixture. The reaction was incubated at 23 ° C for 20 hours. End of the conjugation reaction ("capping") was obtained by adding NaBH4 and the reaction was continued for another 3 hours. The reaction mixture was diluted with a 4-fold volume of 5 mM succinate buffer-0.9% saline, pH 6.0, and then filtered through a 5 pm filter before purification. Concentration and diafiltration of the conjugate were performed using 100K MWCO membranes. Diafiltration was performed against a 40-fold diavolume of 5 mM succinate buffer - 0.9% saline, pH 6.0. The retentate was filtered through 0.45 and 0.22 pm filters and analyzed.
[000233] Various conjugates were obtained using the process described above by varying different parameters (for example, proportion of saccharide-protein intake, reaction concentration, mEq of sodium cyanoborohydride and 0 water content). It was shown that the global data generated from conjugates prepared by the RAC / DMSO process are superior compared to the RAC / aqueous process and allowed to prepare conjugates with good conjugation yield, low% of free saccharides (<5%) and higher degree of conjugation (conjugated lysines). In addition, it was possible to maintain over 80% of all acetyl functionality through the conjugation process with RAC / DMSO. Stability Assessment of Pn-33F RAC / DMSO Glycoconjugates
[000234] Aliquots of representative RAC / DMSO conjugates prepared by the above process were placed in polypropylene tubes which were stored at 4 ° C or 25 ° C and the stability was monitored for 3 months for free saccharides. As shown in Table 10, samples stored at 4 ° C showed an increase in free saccharides of 4.8% in 3 months. However, samples stored at 25 ° C showed an increase of 15.4% in the% of free saccharides in three months. The increase in the% of free saccharides in the RAC conjugates is attributed to the degradation of the conjugate, particularly at 25 ° C. Table 10. Stability Results for RAC / DMSO Conjugates at 4 ° C and 25 ° C
wk = week; M = month.
[000235] The stability of another batch of RAC / DMSO conjugates was also studied at 4 ° C, 25 ° C and 37 ° C. Aliquots were placed in polypropylene tubes and monitored for potential trends in% free saccharides. As shown in Table 11, samples stored at 4 ° C showed a 4.7% increase in the% of free saccharides in 2 months. The increase in free saccharides was significantly greater at 25 ° C and 37 ° C, indicating potential degradation of the conjugate. Table 11. Stability Results for RAC / DMSO Conjugates at 4 ° C, 25 ° C and 37 ° C
wk = week; M = month.
[000236] Even though the conjugates generated by the RAC / DMSO process have preserved the O-acetyl group, the increase in the% of free saccharides observed, particularly at 25 ° C and above, indicated potential instability using this route. In view of this observation of potential instability of the RAC / DMSO conjugates, RAC / DMSO was not considered as ideal for conjugation of the serotype 33F and an alternative chemical pathway was developed to generate more stable conjugates (the eTEC conjugates). Example 8. Preparation of Additional Pn-33F eTEC Conjugates
[000237] Additional Pn-33F and TEC conjugates were generated using the process described in Example 2. The reaction parameters and characterization data for these additional batches of Pn-33F eTEC glycoconjugates are shown in Table 12. Table 12. Parameters Experiments and Characterization Data of Other Pn33F eTEC Conjugates
LOQ = limit of quantification.
[000238] As shown above and in Table 12, several Pn33F conjugates were obtained using the conjugation by eTEC above. The chemistry of eTEC allowed the preparation of conjugates with high productivity, low% of free saccharides and high degree of conjugation (conjugated lysines). In addition, it was possible to maintain more than 80% of the acetyl functionality using the eTEC conjugation process. Example 9. Evaluation of Stability of Pn-33F eTEC Glycoconjugates: Trends in Free Saccharide%
[000239] Aliquots of the 33F-2B conjugate batch (see Table 2) were placed in polypropylene tubes and stored at 4 ° C, 25 ° C and 37 ° C, respectively, and monitored for trends in% free saccharides . The data (% of free saccharides) are shown in Table 13. As shown in this Table, there were no significant changes in free saccharides%. Table 13. Stability of% of free saccharides for glycoconjugates of Pn-33F eTEC at 4 ° C, 25 ° C and 37 ° C
wk = week; M = month.
[000240] The accelerated stability of another batch of conjugate (Lot 33F-3C) was also evaluated at 37 ° C for up to 1 month. As shown in Table 14, there was no significant change in the% of free saccharides at 37 ° C for up to 1 month. Table 14. Stability of% of free saccharides for glycoconjugates of Pn-33F eTEC at 37 ° C

[000241] To further confirm the stability of eTEC conjugates, additional batches of conjugate (33F-3C and 33F-5E (see Table 2 and Table 12)) stored at 4 ° C were monitored for approximately one year for potential trends % of free saccharides. As shown in Table 15, there was no significant change in the% free saccharide levels for conjugates stored at 4 ° C for an extended period of up to about one year. Table 15. Stability Results of% Free Saccharides for Pn-33F eTEC Glycoconjugates at 4 ° C
MN = Month.
[000242] In contrast to the RAC / aqueous and RAC / DMSO conjugates, the 33F serotype conjugates generated by chemistry with 33F and eTEC have been shown to be significantly more stable, with no noticeable degradation, as monitored by trends in free saccharides at various temperatures ( in real time and accelerated).
[000243] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the technique to which the present invention belongs. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[000244] Although the foregoing invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.

权利要求:
Claims (21)
[0001]
1. Method of manufacturing a glycoconjugate comprising a saccharide conjugated to a carrier protein by means of a spacer (2 - ((2-oxoethyl) thio) ethyl) carbamate (eTEC), characterized by the fact that it comprises the steps of: ) reacting a saccharide with 1,1'-carbonyl-di- (1,2,4-triazole) (CDT) or 1, T-carbonyldiimidazole (CDI) in an organic solvent to produce an activated saccharide; b) reaction of the activated saccharide with cystamine or cysteamine or a salt thereof to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reaction of the thiolated saccharide-carrier protein conjugate with (i) N-acetyl-L-cysteine as a first capping reagent; and / or (ii) iodoacetamide as a second capping reagent; where an eTEC-linked glycoconjugate is produced, where the organic solvent in step a) is a polar aprotic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N - methyl-2-pyrrolidone (NMP), acetonitrile, 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone (DMPU) and hexamethylphosphoramide (HMPA) or a mixture thereof, where saccharide is a capsular polysaccharide derived from S. pneumoniae selected from the group consisting of pneumococcal capsular polysaccharides (Pn) of serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, where the carrier protein is CRM197, where the reducing agent in step c) is tris (2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT) or mercaptoethanol.
[0002]
2. Method according to claim 1, characterized by the fact that it further comprises a step of combining the saccharide by means of reaction with triazole or imidazole to provide a combined saccharide, in which the combined saccharide is frozen in wrap, lyophilized and reconstituted in an organic solvent before step a).
[0003]
3. Method according to claim 1 or 2, characterized by the fact that it further comprises purification of the thiolated polysaccharide produced in step c), wherein the purification step comprises diafiltration
[0004]
Method according to any one of claims 1 to 3, characterized in that the carrier protein is activated with an activated derivative of bromoacetic acid.
[0005]
5. Method according to claim 4, characterized by the fact that the bromoacetic acid derivative is the N-hydroxysuccinimide ester of bromoacetic acid (BAANS).
[0006]
Method according to any one of claims 1 to 5, characterized in that it further comprises purification of the linked glycoconjugate by eTEC by means of diafiltration.
[0007]
Method according to any one of claims 1 to 6, characterized in that the saccharide: carrier protein (w / w) ratio is between 0.2 and 4 or between 0.4 and 1.7.
[0008]
Method according to any one of claims 1 to 7, characterized in that the capsular polysaccharide is a Pn capsular polysaccharide of serotype 11A, 10A, 22F or 33F.
[0009]
9. Glycoconjugate, characterized by the fact that it comprises a bacterial capsular polysaccharide conjugated to a carrier protein by means of an eTEC spacer, in which the polysaccharide is covalently linked to the eTEC spacer via a carbamate bond and the carrier protein is covalently linked to the eTEC spacer through an amide bond, in which the capsular polysaccharide is derived from S. pneumoniae and selected from the group consisting of Pn capsular polysaccharides of serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F; where the carrier protein is CRM197.
[0010]
10. Glycoconjugate according to claim 9, characterized in that the capsular polysaccharide is a Pn capsular polysaccharide of serotype 10A, 11A, 22F or 33F.
[0011]
11. Glycoconjugate according to any one of claims 9 to 10, characterized in that the polysaccharide has a molecular weight between 10 kDa and 2,000 kDa, between 50 kDa and 2,000 kDa, between 50 kDa and 20,000 kDa or between 500 kDa and 10,000 kDa.
[0012]
12. Glycoconjugate according to any of claims 9 to 11, characterized in that the polysaccharide has an O-acetylation degree between 75-100%.
[0013]
13. Glycoconjugate according to any one of claims 9 to 12, characterized by the fact that CRM197 comprises 2 to 20 or 4 to 16 lysine residues covalently linked to the polysaccharide through an eTEC spacer.
[0014]
14. Glycoconjugate according to any one of claims 10 to 13, characterized by the fact that the saccharide: carrier protein (w / w) ratio is between 0.2 and 4 or between 0.4 and 1.7.
[0015]
15. Glycoconjugate according to any one of claims 9 to 14, characterized by the fact that at least one link between CRM197 and 0 saccharide occurs every 25, 15, 10 or 4 saccharide repeat units of the polysaccharide.
[0016]
16. Glycoconjugate according to any one of claims 9 to 15, characterized by the fact that it has a molecular weight distribution (Kd) of> 35% to <0.3.
[0017]
17. Immunogenic composition, characterized by the fact that it comprises glycoconjugate, as defined in any of claims 9 to 16, and a pharmaceutically acceptable excipient, vehicle or diluent.
[0018]
18. Immunogenic composition according to claim 17, characterized by the fact that it further comprises an additional antigen.
[0019]
19. Immunogenic composition according to claim 18, characterized in that the additional antigen comprises a glycoconjugate of a capsular polysaccharide selected from the group consisting of Pn capsular polysaccharides of serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F.
[0020]
20. Immunogenic composition according to any one of claims 17 to 19, characterized in that it further comprises an adjuvant.
[0021]
21. Immunogenic composition according to claim 20, characterized in that the adjuvant is an adjuvant based on aluminum selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.

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公开号 | 公开日 | 专利标题

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BR112015003227B1|2020-10-27|a method of making a glycoconjugate comprising a saccharide conjugated to a carrier protein by means of a spacer | thio) ethyl) carbamate |, glycoconjugate and immunogenic composition

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US20200246475A1|2020-08-06|Immunogenic glycoprotein conjugates

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TWI720448B|2021-03-01|Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof

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AU2021206895A1|2021-08-12|Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

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US20210196810A1|2021-07-01|Immunogenic Compositions Comprising Conjugated Capsular Saccharide Antigens and Uses Thereof

同族专利:

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公开号 | 公开日

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CA2881420A1|2014-02-20|

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KR101742406B1|2017-05-31|

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RU2016136630A|2018-12-11|

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BR112015003227A2|2017-07-04|

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MX2015001992A|2015-10-05|

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EP2885007B1|2018-10-10|

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EP2885007A1|2015-06-24|

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TWI480049B|2015-04-11|

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US20170224804A1|2017-08-10|

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HK1210021A1|2016-04-15|

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DK2885007T3|2018-12-03|

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AU2013303826A1|2015-02-12|

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IN2015DN00694A|2015-06-26|

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JP6291495B2|2018-03-14|

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RU2645071C2|2018-02-15|

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US10583187B2|2020-03-10|

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DK3421051T3|2020-06-22|

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US9517274B2|2016-12-13|

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MX363511B|2019-03-26|

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PT2885007T|2018-12-10|

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PT3421051T|2020-06-26|

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CN104661684B|2018-03-20|

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WO2014027302A1|2014-02-20|

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US20150216996A1|2015-08-06|

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CA2881420C|2016-11-15|

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CN104661684A|2015-05-27|

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SI2885007T1|2018-12-31|

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PE20150464A1|2015-04-25|

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IL237066A|2019-03-31|

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SI3421051T1|2020-08-31|

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HUE049531T2|2020-10-28|

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JP2015524839A|2015-08-27|

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AU2013303826B2|2017-06-29|

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ES2700824T3|2019-02-19|

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RU2016136630A3|2019-12-30|

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PL2885007T3|2019-02-28|

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US11110160B2|2021-09-07|

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US9950054B2|2018-04-24|

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US20180221467A1|2018-08-09|

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TW201420115A|2014-06-01|

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RU2724840C2|2020-06-25|

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PL3421051T3|2020-10-05|

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SA515360035B1|2015-09-15|

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EP3421051A1|2019-01-02|

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ES2800479T3|2020-12-30|

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SG11201500566XA|2015-04-29|

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US20200246448A1|2020-08-06|

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PH12015500243A1|2015-03-30|

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EP3421051B1|2020-05-13|

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NZ704490A|2017-07-28|

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PH12015500243B1|2015-03-30|

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MY167579A|2018-09-20|

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KR20150041139A|2015-04-15|

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AR092368A1|2015-04-15|

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HUE041381T2|2019-05-28|

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RU2015103017A|2016-10-10|

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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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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 47/00 (2006.01), A61P 31/04 (2006.01) |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-02-12| 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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2019-03-19| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-09-10| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-04-14| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-27| 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 12/08/2013, 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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US201261684043P| true| 2012-08-16|2012-08-16|

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US61/684,043|2012-08-16|

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PCT/IB2013/056597|WO2014027302A1|2012-08-16|2013-08-12|Glycoconjugation processes and compositions|

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