Conjugation of saccharide antigens using acetoxyborohydrides

Abstract

The present disclosure relates to an improved method for producing glycoconjugates, which involves using a reducing mixture containing in situ prepared acetoxyborohydrides to conjugate saccharides to carrier proteins.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing glycoconjugates by conjugating polysaccharides to carrier proteins using acetoxyborohydride prepared in situ via reductive amination. [Background technology]

[0002] Bacterial capsular polysaccharides are long polymers composed of many repeating units of simple sugars that protect bacteria from phagocytosis. Antibodies against the capsular polysaccharides of many pathogenic bacteria can stimulate immune responses by enhancing bacterial phagocytosis. However, vaccines made from purified polysaccharides, while partially immunogenic in adults, do not elicit antibody responses in infants or children. This problem can be overcome by chemically conjugating polysaccharides to carrier proteins, thereby enhancing their immunogenicity. Conjugation converts polysaccharides, which are T-cell-independent antigens, into proteins, which are T-cell-dependent antigens, enabling isotype switching, affinity maturation, and memory B cell formation.

[0003] The two most widely used methods for conjugating bacterial polysaccharides to carrier proteins are amidation and reductive amination. In amidation, the reducing end of the polysaccharide is first oxidized to the corresponding aldonic acid. This step is followed by reaction of the acid group of the aldonic acid with a primary amine on the lysine side chain of the carrier protein. In reductive amination, the reducing end of the polysaccharide is first oxidized to the corresponding aldehyde. This reaction is followed by formation of a Schiff base between the aldehyde group and the primary amine (or N-terminal amine) on the lysine side chain of the carrier protein, generating an imine, which is then reduced with a hydride source. Cyanoborohydride has been used as a hydride source for the conjugation of bacterial polysaccharides, such as Streptococcus pneumoniae saccharides (WO 1987 / 006838). However, the use of cyanoborohydride has drawbacks such as a slow reaction time and contamination of the produced amine with cyanide (Ahmed F. et al. J. Org. Chem., 1996, 61:3849-3862).

[0004] Triacetoxyborohydride has been used as an alternative to cyanoborohydride in the reduction of aldehydes and ketones (Ahmed F. et al. J. Org. Chem., 1996, 61:3849-3862) and carbohydrates (Dalpathado et al. Anal. Bioanal. Chem. (2005) 281:130-1137). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO1987 / 006838 [Non-patent literature]

[0006] [Non-Patent Document 1] Ahmed F. et al. J. Org. Chem., 1996, 61: 3849-3862). [Non-patent document 2] Dalpathado et al. Anal. Bioanal. Chem. (2005) 281:130-1137. Summary of the Invention [Problem to be solved by the invention]

[0007] Triacetoxyborohydride has also been used to reduce imines formed during the conjugation of capsular polysaccharides to protein carriers (EP 2683408). However, the efficiency of triacetoxyborohydride-mediated reductive amination needs to be improved. [Means for solving the problem]

[0008] Thus, the present invention provides an improved method for conjugating saccharide antigens to carrier proteins using acetoxyborohydrides, which involves using a reducing mixture containing freshly or in situ prepared acetoxyborohydrides for reductive amination of the Schiff base formed between the saccharide and the carrier protein upon conjugation.

[0009] In one aspect, the present disclosure provides a method for conjugating an antigen to a carrier protein. The method includes: (a) activating an antigen to form an activated antigen; (b) reacting the activated antigen with a carrier protein to obtain an intermediate, in which the activated antigen and the carrier protein are conjugated through an imine group; and (c) reducing the imine group by a process including: (i) mixing acetic acid with a borohydride solution to prepare a reducing mixture containing acetoxyborohydride; and (ii) treating the intermediate with the reducing mixture. At least about 20% of the acetoxyborohydrides in the reducing mixture used to treat the intermediate are diacetoxyborohydride (DAB). The antigen is a saccharide. Alternatively, the reducing mixture is referred to herein as an in situ acetoxyborohydride reducing mixture.

[0010] In some embodiments, the remainder of the acetoxyborohydride in the reducing mixture is either triacetoxyborohydride (TAB) or a mixture of TAB and monoacetoxyborohydride (MAB).

[0011] In some embodiments, the saccharide is a bacterial capsular polysaccharide.

[0012] In some embodiments, the reducing mixture is held (i.e., not used for a period of time after preparation of the reducing mixture), such as at least about 30 minutes, about 1 to about 8 hours, or about 2 to about 6 hours, before being used to process the intermediate.

[0013] In some embodiments, the borohydride used is sodium borohydride or potassium borohydride. In some embodiments, a borohydride compound solution is prepared by dissolving it in dimethyl sulfoxide (DMSO).

[0014] In some embodiments, step (c)(i) is carried out at a temperature of about 20°C to about 35°C, about 20°C to about 30°C, or about 20°C to about 25°C.

[0015] In some embodiments, unreacted carbonyl groups (e.g., residual aldehyde groups) are reduced by a reducing mixture. In some embodiments, the method further comprises reducing the unreacted carbonyl groups using a borohydride compound (e.g., sodium borohydride or potassium borohydride). In some embodiments, the reducing mixture comprises at least about 30% diacetoxyborohydride.

[0016] In some embodiments, the bacterial capsular polysaccharide conjugated by the above methods is derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella vi, or Staphylococcus epidermidis.

[0017] In some embodiments, the carrier protein can be tetanus toxoid (TT), tetanus toxoid fragment C, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD (pneumococcal histidine triad protein D), PhtDE, or N19. In some embodiments, the carrier protein is CRM197, and the saccharide is conjugated to a lysine residue of CRM197 to yield a molar ratio of conjugated CRM197 lysine residues to total CRM197 amine residues of about 0.5:10 to about 5:10.

[0018] In some embodiments, the conjugated antigen produced according to the methods of the present invention has a molecular weight of about 50 kDa to about 20,000 kDa, hi some embodiments, the conjugated antigen has less than about 45% free bacterial capsular polysaccharides compared to the total amount of bacterial capsular polysaccharides. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 is a graph showing the concentration variation of acetoxyborohydrides, SMAB (sodium monoacetoxyborohydride), SDAB (sodium diacetoxyborohydride), and STAB (sodium triacetoxyborohydride), in an in situ acetoxyborohydride reducing mixture over time. Figure 1 also shows the relative amounts of SDAB and STAB present in a commercially available STAB. [Figure 2] FIG. 1 shows the variation in size of the conjugates formed as a function of the time the in situ acetoxyborohydride reducing mixture is held before use for the reduction of the imine group in the conjugation reaction. [Figure 3] Figure 1 shows the effect of the temperature at which the in situ acetoxyborohydride reducing mixture is prepared on the conjugation efficiency, as measured by the size of the resulting conjugate (n=3). [Figure 4] Figure 4 shows functional antibody (OPA) titers in mice immunized with pneumococcal serotypes 9N, 22F, and 35B. These serotypes were conjugated with (Arm 2) or without (Arm 1) an in situ acetoxyborohydride reducing mixture. In Arm 1, cyanoborohydride was used as the reducing agent for serotypes 9N and 22F, and no reducing agent was used for serotype 35B (Figures 4A, 4B, and 4C). DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure may be understood more readily by reference to the following detailed description of various embodiments of the disclosure and the examples contained herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. In describing the embodiments and in the claims, certain terms will be used in accordance with the definitions set forth below.

[0021] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0022] As used herein, the term "about" means within a statistically significant range of values, such as a specified concentration range, time frame, molecular weight, temperature, or pH. Such a range can be within one order of magnitude, usually within 10%, and more typically within 5% or 1% of a given value or range. In some cases, such a range can be within the range of experimental error typical of the standard method used to measure and / or determine the given value or range. The allowable variation encompassed by the term "about" will depend on the particular system under investigation and will be readily apparent to one of ordinary skill in the art. Whenever a range is recited herein, each of the endpoints of the range, as well as all values ​​within the range, are contemplated as embodiments of the present disclosure.

[0023] It should be noted that in this disclosure, the terms "comprises," "comprised," "comprising," "contains," "containing," and the like mean "includes," "included," "including," etc. Such terms refer to the inclusion of a particular component or set of components without the exclusion of other components.

[0024] Terms such as "consisting essentially of" and "consists essentially of" permit the inclusion of additional components or steps that do not detract from the novel and basic characteristics of the disclosure. That is, they exclude additional, unrecited components or steps that detract from the novel and basic characteristics of the disclosure. The terms "consist of" and "consisting of" are limiting terms. Thus, these terms imply the inclusion of a particular component or set of components and the exclusion of all other components.

[0025] The term "saccharide" is used herein to refer to a polysaccharide, oligosaccharide, or monosaccharide.

[0026] The term "conjugate" or "glycoconjugate" as used herein refers to a saccharide covalently conjugated to a carrier protein. The glycoconjugates of the present disclosure and immunogenic compositions comprising them may contain small amounts of free saccharide.

[0027] As used herein, the term "free saccharide" refers to a saccharide that is not covalently conjugated to a carrier protein, but is nevertheless present in a glycoconjugate composition. Free saccharides can be non-covalently associated with (i.e., non-covalently bound, adsorbed, or entrapped within) a conjugated saccharide-carrier protein glycoconjugate. The terms "free polysaccharide" and "free capsular polysaccharide" are used herein to convey the same meaning with respect to carbohydrate conjugates in which the saccharide is a polysaccharide or capsular polysaccharide, respectively.

[0028] As used herein, "fresh or in situ prepared acetoxyborohydride" refers to a mixture of acetoxyborohydrides, including diacetoxyborohydride, triacetoxyborohydride, and optionally monoacetoxyborohydride, prepared by combining a borohydride (e.g., sodium borohydride) with acetic acid less than 25 hours before use in a conjugation reaction. For example, the acetoxyborohydrides can be prepared 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes before use.

[0029] As used herein, "to conjugate," "conjugated," and "conjugating" refer to a process in which a saccharide, such as a bacterial capsular polysaccharide, is covalently attached to a carrier molecule or protein. Conjugation can be carried out according to the methods described below or by other processes known in the art. Conjugation enhances the immunogenicity of the bacterial capsular polysaccharide.

[0030] The term "subject" refers to any mammal, including humans, or any bird, fish, reptile, amphibian, or other animal. The term "subject" also includes domestic pets and research animals. Non-limiting examples of domestic pets and research animals include dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, monkeys, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes domestic animals. Non-limiting examples of domestic animals include alpacas, bison, camels, cows, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0031] Complex carbohydrates The present invention relates to a method for conjugating saccharides to carrier proteins, i.e., to a method for producing glycoconjugates by reducing imine groups formed during the conjugation process using a freshly or in situ prepared reducing mixture comprising diacetoxyborohydrides, triacetoxyborohydrides, and optionally monoacetoxyborohydrides.

[0032] In the glycoconjugates of the present disclosure, the saccharide is a monosaccharide, oligosaccharide, or polysaccharide, and the carrier protein can be any suitable carrier protein further described herein or known to one of skill in the art. In some embodiments, the saccharide is a polysaccharide, particularly a bacterial capsular polysaccharide such as that from Streptococcus pneumoniae (S. pneumoniae, serotype 35B). In some embodiments, the carrier protein is CRM197 (cross-reactive substance 197 from Corynebacterium diphtheriae C7).

[0033] Capsular polysaccharides can be produced from various serotypes, such as serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F of Streptococcus pneumoniae (S. pneumoniae), by standard techniques known to those skilled in the art. Conjugates can be prepared in separate processes and formulated into single-dose formulations. For example, each pneumococcal polysaccharide serotype can be grown separately, and then the individual polysaccharides can be purified by steps including one or more of centrifugation, precipitation, ultrafiltration, and column chromatography. Purified polysaccharides can be chemically activated to allow the saccharides to react with carrier proteins. Once activated, each capsular polysaccharide can be separately conjugated to a carrier protein to form a glycoconjugate. Each capsular polysaccharide in the formulation may be conjugated to the same carrier protein. Alternatively, multiple carrier proteins may be used for polysaccharide conjugation. Chemical activation of the polysaccharides can be carried out by conventional means. See, e.g., U.S. Patent Nos. 4,902,506, 7,709,001, and 7,955,605.

[0034] In one embodiment, the glycoconjugates of the present disclosure have a molecular weight of about 50 kDa to about 20,000 kDa. In another embodiment, the glycoconjugates have a molecular weight of about 200 kDa to about 10,000 kDa. In another embodiment, the glycoconjugates have a molecular weight of about 500 kDa to about 5,000 kDa. In one embodiment, the glycoconjugates have a molecular weight of about 1,000 kDa to about 3,000 kDa. In other embodiments, the glycoconjugates have a molecular weight of about 600 kDa to about 2800 kDa; about 700 kDa to about 2700 kDa; about 1000 kDa to about 2000 kDa; about 1800 kDa to about 2500 kDa; about 1100 kDa to about 2200 kDa; about 1900 kDa to about 2700 kDa; about 1200 kDa to about 2400 kDa; about 1700 kDa to about 2600 kDa; about 1300 kDa to about 2600 kDa; or about 1600 kDa to about 3000 kDa. Any integer within the above ranges is contemplated as an embodiment of the present disclosure.

[0035] In one embodiment, the polysaccharide is a capsular polysaccharide from Neisseria meningitidis (N. meningitidis). In some such embodiments, the capsular polysaccharide is selected from the group consisting of serotype A, B, C, W135, X, and Y capsular polysaccharides of N. meningitidis.

[0036] In one embodiment, the polysaccharide is a capsular polysaccharide from Streptococcus pneumoniae (S. pneumoniae). For example, the capsular polysaccharide is from Streptococcus pneumoniae (S. pneumoniae) serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, DeOAc15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 23B, 24F, 31, 24F, 31, 33F, or 35B.

[0037] As used herein, DeOAc15B (de-O-acetylated serotype) refers to a pneumococcal polysaccharide that is substantially equivalent to serotype 15C pneumococcal polysaccharide and has a substantially identical NMR spectrum (data not shown). As used herein, de-O-acetylated serotype 15B pneumococcal polysaccharide and serotype 15C pneumococcal polysaccharide can each have an O-acetyl content per repeat unit in the range of 0-5%, 0-4%, 0-3%, 0-2%, 0-1%, 0-0.5%, or 0-0.1%, or can contain no O-acetyl at all. Spencer BL et al. reported that pneumococcal polysaccharide 15C may be slightly O-acetylated (Spencer, BL et al., Clin. Vac. Immuno. (2017) 24(8):1-13). Thus, in any of the embodiments of the methods described herein, de-O-acetylated serotype 15B (DeOAc15B) can be used in place of serotype 15C. The process of de-O-acetylation is known in the art, for example, as described in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9):1223-1227.

[0038] In some embodiments, the glycoconjugates of the present disclosure comprise bacterial capsular polysaccharides, wherein the capsular polysaccharides have a molecular weight of 10 kDa to 2,000 kDa or 50 kDa to 1,000 kDa.

[0039] Glycoconjugates comprising capsular polysaccharides covalently conjugated to carrier proteins produced by the methods of the present invention can have one or more of the following characteristics: the polysaccharide molecular weight is between 50 kDa and 1,000 kDa; the glycoconjugate molecular weight is between 1,000 kDa and 5,000 kDa; and the conjugate comprises less than about 45% free polysaccharide. In some embodiments, the polysaccharide has a molecular weight between 10 kDa and 2,000 kDa. In some embodiments, the glycoconjugate has a molecular weight between 50 kDa and 20,000 kDa. In other embodiments, the glycoconjugate has a molecular weight between 200 kDa and 10,000 kDa. In other embodiments, the conjugate comprises less than about 30%, 20%, 15%, 10%, or 5% free polysaccharide relative to the total polysaccharide. The amount of free polysaccharide can be measured as a function of time, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 120 days or even longer after the conjugate is made.

[0040] The number of lysine residues in a carrier protein conjugated to a saccharide can be characterized as a range of conjugated lysine residues, which can be expressed as a molar ratio. The carrier protein can be CRM197, which contains 39 lysine amines. All of these amines, as well as the N-terminal amine (i.e., a total of 40 amine residues), can serve as sites for conjugation. However, not all of these amine residues are equally reactive. In some embodiments, in glycoconjugates made with CRM197, only 4-15 lysine residues are covalently linked to the saccharide, resulting in a molar ratio of conjugated lysine residues to total CRM197 amine residues of about 1:10 to about 4:10. In other embodiments, 2-20 lysine residues of CRM197 are covalently linked to the saccharide, resulting in a molar ratio of conjugated lysine residues to CRM197 amine residues of about 0.5:10 to about 5:10.

[0041] The saccharide:carrier protein ratio (by weight) can also vary. In one embodiment, the saccharide:carrier protein ratio (by weight) is 0.2 to 4. In another embodiment, the saccharide:carrier protein ratio (by weight) is 1.1 to 1.7. In some embodiments, the saccharide is a bacterial capsular polysaccharide and the saccharide:carrier protein ratio (by weight) is 0.2 to 4. In other embodiments, the saccharide is a bacterial capsular polysaccharide and the saccharide:carrier protein ratio (by weight) is 1.1 to 1.7. In some such embodiments, the carrier protein is CRM197.

[0042] The frequency of attachment of saccharide chains to lysine residues on the carrier protein can also vary. For example, in one embodiment, there is at least one covalent linkage between the carrier protein and the polysaccharide for every 100 saccharide repeat units of the polysaccharide. In one embodiment, there is at least one covalent linkage between the carrier protein and the polysaccharide for every 50 saccharide repeat units of the polysaccharide. In one embodiment, there is at least one covalent linkage between the carrier protein and the polysaccharide for every 25 saccharide repeat units of the polysaccharide. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every four saccharide repeat units of the polysaccharide. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeat units of the polysaccharide. In a further embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeat units of the polysaccharide. In another embodiment, at least one linkage between the carrier protein and the saccharide occurs at 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.

[0043] In some embodiments, the carrier protein is CRM197 and the covalent linkage between CRM197 and the polysaccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide, hi some such embodiments, the polysaccharide is a bacterial capsular polysaccharide, such as a capsular polysaccharide from Streptococcus pneumoniae (S. pneumoniae) or Neisseria meningitidis (N. meningitidis).

[0044] In one embodiment, the complex carbohydrates contain less than about 45% free sugars relative to the total amount of sugars. In another embodiment, the complex carbohydrates contain less than about 30% free sugars relative to the total amount of sugars. In another embodiment, the complex carbohydrates contain less than about 20% free sugars relative to the total amount of sugars. In a further embodiment, the complex carbohydrates contain less than about 10% free sugars relative to the total amount of sugars. In another embodiment, the complex carbohydrates contain less than about 5% free sugars relative to the total amount of sugars.

[0045] In some embodiments, the glycoconjugates comprise less than about 30 mol%, less than about 25 mol%, less than about 20 mol%, less than about 15 mol%, or less than about 10 mol% carrier protein residues relative to the total amount of glycoconjugates.

[0046] Carrier proteins "Protein carrier" or "carrier protein" or "carrier" refers to a protein molecule that can be conjugated to an antigen (such as a capsular polysaccharide) against which an immune response is desired.

[0047] Conjugation to a carrier can enhance the immunogenicity of an antigen. Protein carriers for antigens can be toxins, toxoids, or cross-reactive mutants (CRMs) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, staphylococci, and streptococci. In one embodiment, the carrier is diphtheria toxoid CRM.sub.197, derived from Corynebacterium diphtheriae (C. diphtheriae) strain C7(6197), which produces the CRM.sub.197 protein. This strain has ATCC deposit number 53281. Methods for producing CRM.sub.197 are described in U.S. Patent No. 5,614,382, the entire contents of which are incorporated herein by reference. Alternatively, fragments or epitopes of protein carriers or other immunogenic proteins can be used. For example, the hapten antigen can be conjugated to a T cell epitope of a bacterial toxin, toxoid, or CRM. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in International Patent Application Publication No. WO 2004 / 083251), Escherichia coli (E. coli) LT, Escherichia coli (E. coli) ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane conjugate c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), or Haemophilus influenzae protein D can also be used. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.

[0048] Characterization of polysaccharide-protein conjugates Glycoconjugates can be characterized based on their molecular weight, which can be either the number-average molecular weight (Mn) or the weight-average molecular weight (Mw). Glycoconjugates are polymers that exist as a distribution of chain lengths and molecular weights. Therefore, it is impossible to know the exact molecular weight of a glycoconjugate. Instead, averages of various parameters are used to indicate its molecular weight. Mn and Mw are two such averages. Mn is the statistical average molecular weight of all polymer chains in a sample. It is calculated by dividing the total weight of the polymer by the total number of molecules. On the other hand, Mw takes into account the molecular weight of the polymer chain. This is based on the fact that molecules with higher masses account for more of the total mass of a polymer sample than molecules with lower masses. Therefore, Mw is an average weight and reflects the average weight of molecules in a polymer sample. Various methods are used to determine Mn and Mw. For glycoconjugates, Mn can be measured by size exclusion chromatography, and Mw can be measured using static light scattering. Both Mn and Mw can be measured using simultaneous size exclusion chromatography (SEC) followed by sequential monitoring of refractive index (RI), ultraviolet absorbance (UV), and multi-angle laser light scattering (MALS). Pollock JF et al., Bioconjug Chem. 2012 September 19;23(9):1794-1801.

[0049] Glycoconjugates are also characterized by the number of lysine residues in the carrier protein that are conjugated to saccharides. This number can be expressed as conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein by covalent attachment to a polysaccharide can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation reduces the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugated substance. For example, the degree of conjugation can be as low as 2 or as high as 15.

[0050] Another method of characterizing glycoconjugates is by using the carbohydrate to carrier protein (wt / wt) ratio (Ps:Pr). For example, Ps:Pr can be as low as 0.5 or as high as 3.0. Glycoconjugates may include free carbohydrates that are not covalently bound to a carrier protein but are still present in the glycoconjugate composition. Free carbohydrates may be in glycoconjugates that are non-covalently associated with (i.e., non-covalently bound, adsorbed, or entrapped within or on) the glycoconjugate. For example, glycoconjugates may contain less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharides compared to the total amount of polysaccharides.

[0051] Conjugation To conjugate with a carrier protein, polysaccharides must be activated (chemically modified) and then chemically linked to a carrier, such as a protein. Prior to the activation step, saccharides can be hydrolyzed or mechanically sized by pressure homogenization to achieve a molecular weight (e.g., 50 kDa–500 kDa) suitable for activation and subsequent conjugation. Partial oxidation of carbohydrates in polysaccharides can be effectively used to generate aldehyde groups, which can then couple to amine groups, such as lysine residues, on carrier proteins to produce immunogenic conjugates. It is important that the method used to conjugate polysaccharides to carrier proteins yields stable covalent bonds and that the reaction conditions are mild enough to maintain the structural integrity of the individual components. Commonly used methods for activating polysaccharides and conjugating them to carrier proteins include reductive amination chemistry (RAC), cyanylation, and the use of carbodiimides. Reductive amination typically involves the selective oxidation of adjacent -OH groups to active aldehyde groups using sodium or potassium periodate or periodic acid. Cyanylation is used to randomly convert -OH groups to active -CN groups. Carbodiimides are used to activate carboxyl groups by displacing the -OH group with the carbodiimide.

[0052] Reductive amination chemistry (RAC) is one of the most commonly used methods for coupling polysaccharides to proteins, since the reaction between the resulting carbonyl groups of the polysaccharide and the amino groups of the carrier protein can form the corresponding Schiff base, which can be selectively reduced in the presence of sodium cyanoborohydride to yield a stable saturated carbon-nitrogen bond. Furthermore, reductive amination can be performed in aqueous solution under conditions mild enough to maintain the structural integrity of the saccharide and protein components. After conjugation, unreacted aldehydes can be reduced with sodium borohydride. The conjugate can then be purified, for example, by ultrafiltration / diafiltration.

[0053] However, as described in EP 2683408B1, the use of cyanoborohydride ions has certain drawbacks, such as relatively slow reaction times and the potential for contamination of the cyanide conjugate (Ahmed F. et al., J. Org. Chem., 1996, 61:3849-3862). The use of microwave irradiation has been proposed as a solution to the slow reaction time (EP 1035137). EP 2683408B1 also proposes the use of triacetoxyborohydride (BH(OA)3) as an alternative to cyanoborohydride, because the reductive amination using triacetoxyborohydride anions is faster than the equivalent reaction using cyanoborohydride ions and does not produce toxic by-products. The proposed use of triacetoxyborohydride in the conjugation of bacterial capsular polysaccharides is based on previous reports of its use in the reductive amination of aldehydes and ketones (J. Org. Chem., 1996, 61:3849-3862) and carbohydrates (Dalpathado et al., Anal. Bioanal. Chem. (2005) 281:130-1137).

[0054] However, the present inventors found that the reductive amination process using commercially available triacetoxyborohydride was not very effective and identified room for improvement. This improvement involved performing the reductive amination using a freshly prepared acetoxyborohydride-containing reducing mixture. It was observed that the reducing mixture prepared by combining sodium borohydride and acetic acid, when fresh (within approximately 15 hours of preparation), contained a higher concentration of diacetoxyborohydride (DAB) than the commercially available triacetoxyborohydride reagent. See Figure 1. It was also observed that the concentration of DAB in the reducing mixture decreased over time (Figure 1), and this decrease paralleled the decrease in the size of the resulting conjugates. See Figure 2. In other words, reducing mixtures containing higher concentrations of DAB produced a greater amount of larger conjugates. It is noteworthy that among MAB, DAB, and TAB, MAB had the highest reduction potential, followed by DAB, and TAB had the lowest reduction potential. Improved conjugation and conjugation properties are observed when using in situ prepared acetoxyborohydride reducible mixtures compared to commercially available TAB, because the in situ prepared acetoxyborohydride reducible mixtures contain a relatively higher proportion of acetoxyborohydride species with higher reduction potentials (Gordon W, Gribble, Chem. Soc. Rev., 1998, 27:395-404).

[0055] It has also been found that holding the reaction mixture for a period of time, e.g., 1 hour, before using it in the conjugation reaction results in consistent conjugate size and consistency across production. Various aspects of conjugating saccharide antigens using the freshly prepared acetoxyborohydride reducing mixture described herein are described in more detail in the following numbered embodiments.

[0056] Embodiment 1 is a method of conjugating an antigen to a carrier protein, comprising: a) activating an antigen to form an activated antigen; b) reacting the activated antigen with a carrier protein to obtain an intermediate in which the activated antigen and the carrier protein are conjugated via an imine group; and c) (i) combining acetic acid with a borohydride solution to form a reducing mixture comprising acetoxyborohydride; and (ii) treating the intermediate with the reducing mixture, wherein at least about 20% of the acetoxyborohydrides in the reducing mixture are diacetoxyborohydrides; reducing the imine group by a process comprising: thereby producing a conjugate antigen, wherein the antigen is a saccharide. A method is provided.

[0057] Embodiment 2 provides the method of embodiment 1, wherein in step (ii), the remainder of the acetoxyborohydrides in the reducing mixture is either triacetoxyborohydride or a mixture of triacetoxyborohydride and monoacetoxyborohydride.

[0058] Embodiment 3 provides the method of embodiment 1 or 2, wherein the saccharide is a bacterial capsular polysaccharide.

[0059] Embodiment 4 provides the method of any of Embodiments 1-3, wherein the reducing mixture is held for a period of at least about 30 minutes before treating the intermediate with the reducing mixture.

[0060] Embodiment 5 provides the method of any of embodiments 1-4, wherein the borohydride is sodium borohydride or potassium borohydride.

[0061] Embodiment 6 provides the method of any of Embodiments 1-5, wherein the borohydride solution is prepared by dissolving the borohydride in dimethyl sulfoxide (DMSO). Alternatively, a non-aqueous solvent such as acetonitrile, 1,2-dimethoxyethane, or other suitable non-aqueous solvent known to one of skill in the art can be used.

[0062] Embodiment 7 provides the method of any of Embodiments 1-6, wherein step (c)(i) is carried out at a temperature of about 20°C to about 35°C.

[0063] Embodiment 8 provides the method of embodiment 7, wherein step (c)(i) is carried out at a temperature of about 20°C to about 25°C.

[0064] Embodiment 9 provides the method of any of embodiments 1-8, wherein unreacted carbonyl groups (e.g., residual aldehyde groups) are reduced with the reducing mixture.

[0065] Embodiment 10 provides the method of any of embodiments 1-8, further comprising reducing unreacted carbonyl groups using a borohydride, such as sodium borohydride or potassium borohydride.

[0066] Embodiment 11 provides the method of any of embodiments 1-10, wherein the reducing mixture is held for about 1 to about 8 hours before use.

[0067] Embodiment 12 provides the method of embodiment 11, wherein the reducing mixture is held for about 2 to about 6 hours before use.

[0068] Embodiment 13 provides the method of any of embodiments 1-12, wherein the reducing mixture comprises at least about 25% diacetoxyborohydride or at least about 30% diacetoxyborohydride.

[0069] Embodiment 14 provides the method of any of embodiments 1-13, wherein the bacterial capsular polysaccharide is from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella vi, or Staphylococcus epidermidis.

[0070] Embodiment 15 provides the method of embodiment 14, wherein the bacterial capsular polysaccharide is from Streptococcus pneumoniae (S. pneumoniae).

[0071] Embodiment 16 provides the method of Embodiment 15, wherein the S. pneumoniae capsular polysaccharide is of a serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, deOAc-15B, 16F, 17F, 18C, 19A, 19F, 20A, 22F, 23A, 23F, 23B, 24F, 31, 24F, 31, 33F, and 35B.

[0072] Embodiment 17 provides the method of embodiment 14, wherein the bacterial capsular polysaccharide is from Neisseria meningitidis (N. meningitidis).

[0073] Embodiment 18 provides the method of embodiment 17, wherein the N. meningitidis capsular polysaccharide is of a serotype selected from the group consisting of A, B, C, W135, X, and Y.

[0074] Embodiment 19 provides the method of any of Embodiments 1 to 18, wherein the carrier protein is a protein selected from the group consisting of tetanus toxoid (TT), tetanus toxoid fragment C, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD (pneumococcal histidine triad protein D), PhtDE, and N19.

[0075] Embodiment 20 provides the method of embodiment 19, wherein the carrier protein is CRM197.

[0076] Embodiment 21 provides the method of embodiment 20, wherein the saccharide is conjugated to a lysine residue of CRM197, resulting in a molar ratio of conjugated CRM197 lysine residues to total CRM197 amine residues of about 0.5:10 to about 5:10.

[0077] Embodiment 22 provides the method of any of embodiments 1 to 21, wherein the conjugated antigen has a molecular weight of about 50 kDa to about 20,000 kDa.

[0078] Embodiment 23 provides the method of any of embodiments 1-22, wherein the conjugated antigen comprises less than about 45% free bacterial capsular polysaccharide compared to the total amount of bacterial capsular polysaccharide.

[0079] Pharmaceutical Composition In another aspect, the present disclosure provides an immunogenic composition comprising a glycoconjugate of the present disclosure and at least one of an adjuvant, a diluent, or a carrier.

[0080] In some embodiments, the present disclosure provides immunogenic compositions comprising a glycoconjugate of the present disclosure and at least one of an adjuvant, a diluent, or a carrier, wherein the glycoconjugate comprises a bacterial capsular polysaccharide covalently conjugated to a carrier protein. In some such embodiments, the capsular polysaccharide is from Streptococcus pneumoniae (S. pneumoniae) or Neisseria meningitidis (N. meningitidis).

[0081] In some embodiments, the immunogenic composition comprises an adjuvant. In some such 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 adjuvant aluminum phosphate.

[0082] In some embodiments, the immunogenic composition comprises a conjugated antigen or a mixture of conjugated antigens, one or more of which are prepared according to the method of any of numbered embodiments 1-23 above, and mixed with a pharmaceutically acceptable excipient.

[0083] In some embodiments, the glycoconjugates or immunogenic compositions of the present disclosure can be used to generate antibodies that function as measured by killing bacteria in animal efficacy models or via opsonophagocytic cell killing assays.

[0084] In another aspect, the present disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition described herein. In a related embodiment, the present disclosure provides a method of inducing an immune response in a subject against a pathogenic bacterium, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition described herein.

[0085] In another aspect, the present disclosure provides a method for preventing or ameliorating a disease or condition caused by a pathogenic bacterium in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition described herein.

[0086] In another aspect, the present disclosure provides a method for reducing the severity of at least one symptom of a disease or condition caused by infection with a pathogenic bacteria in a subject, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition described herein.

[0087] In some embodiments, the pathogenic bacterium is Streptococcus pneumoniae (S. pneumoniae) or Neisseria meningitidis (N. meningitidis). In one embodiment, the pathogenic bacterium is Streptococcus pneumoniae (S. pneumoniae) and the capsular polysaccharide is of a serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, DeOAc15B, 16F, 17F, 18C, 19A, 19F, 20, 22F, 23A, 23F, 23B, 24F, 31, 24F, 31, 33F, and 35B. In one embodiment, the pathogenic bacterium is N. meningitidis and the capsular polysaccharide is selected from the group consisting of N. meningitidis serotype A, B, C, W135, X and Y capsular polysaccharides.

[0088] The present invention will be more clearly understood in light of the following examples which serve to illustrate the invention without, however, limiting its scope. [Example]

[0089] Example 1: In situ acetoxylation Hydrogenation Preparation of boron reducing mixture An acetoxyborohydride reducing mixture was prepared by adding 0.4M to 0.8M sodium borohydride to DMSO and mixing until the sodium borohydride was completely dissolved. Approximately 3 to 5 molar equivalents of acetic acid were then added to the sodium borohydride solution to form the acetoxyborohydride reducing mixture. The reducing mixture contained three reducing species: MAB, DAB, and TAB. These species differ in their reduction potentials. Furthermore, the concentration of each species in the mixture varied with time and temperature.

[0090] The preparation of the acetoxyborohydride reducing mixture is exothermic. The temperature increase during the preparation of the reducing mixture was controlled by adding acetic acid in multiple portions (4–8 additions). The preparation was performed in a glove box to prevent moisture contamination. Moisture induces hydrolysis of the reducing species, further increasing the reaction temperature. Once the preparation was complete, the reducing mixture was held (i.e., allowed to settle) for approximately 2–8 hours before use in the conjugation reaction. It was observed that holding the mixture for the same amount of time before use ensured that the DAB concentration in the mixture was approximately the same for each preparation of the reducing mixture.

[0091] Example 2: In situ acetoxylation Hydrogenation Composition of boron reducing mixture The composition of the in situ acetoxyborohydride reducing mixture is time-dependent. The amounts of the components MAB, DAB, and TAB in the mixture vary over time. These amounts can be measured by NMR. In this example, measurements of the reducing mixture's composition began after the final addition of acetic acid to the borohydride solution. MAB is the most unstable species in the reducing mixture. MAB is consumed within the first two hours of the reaction (see Figure 1). The concentration of DAB in the reducing mixture decreases over time, but the rate of decrease slows. This indicates that the DAB concentration eventually reaches a plateau. On the other hand, the concentration of TAB increases over time as MAB and DAB are converted to TAB.

[0092] Example 3: In situ acetoxylation Hydrogenation Correlation between conjugate size and DAB concentration in boron-reducing mixtures A study was conducted to determine whether there was a correlation between the concentration of DAB in the acetoxyborohydride reducing mixture and the size of the conjugates. Conjugate size reflects the efficiency of the conjugation process. The larger the conjugate size, the more efficient the process. In one study, using the bacterial capsular polysaccharide ST-35B as the antigen, it was observed that as the concentration of DAB in the reducing mixture decreased over time, the size of the ST-35B conjugates also decreased (see Figure 2). The observations were conducted over a 25-hour period. The fact that the amount of TAB in the reducing mixture increased over this period, while the amount of DAB decreased, strongly suggests that the majority of the reductive amination occurring during conjugation is due to reduction by DAB, not TAB. In other words, DAB, not TAB, is the predominant reducing species. It is important to note that MAB levels reach zero within approximately 1–2 h of reducing mixture preparation.

[0093] Conjugation of several S. pneumoniae serotypes was performed using the in situ acetoxyborohydride reducing mixture. These S. pneumoniae serotypes and the conjugation results are shown in Table 1 below.

[0094] Table 1 [Table 1]

[0095] Example 4: In situ acetoxylation Hydrogenation Comparison of Boron Reducing Mixtures with Commercial STAB The concentrations of MAB, DAB, and TAB in the in situ DAB-reducing mixture were compared with the concentrations of these species in a commercially available STAB preparation. As shown in Figure 1, one of the commercially available STAB preparations examined contained approximately 17% DAB and 83% TAB, respectively. The absence of MAB in the commercially available STAB preparation is expected, given the low stability of MAB, which converts to TAB in approximately 1-2 hours.

[0096] Given that DAB is the predominant reducing species (see Example 3) and that DAB is present in the in situ acetoxyborohydride reducible mixture at higher levels than commercial STAB (at least 25 h after preparation, see Figure 1), the in situ acetoxyborohydride reducible mixture is a superior reducing agent to commercial STAB. This was confirmed by directly comparing the conjugate sizes obtained using the in situ acetoxyborohydride reducible mixture described herein with those obtained using commercial STAB (see Table 2). For the comparison, the same molar equivalents of commercial acetoxyborohydride and in situ acetoxyborohydride were used. Results obtained using ST-35B, ST-9N, and ST-22F as bacterial polysaccharides are shown in Table 2 below.

[0097] Table 2 [Table 2] The results show that the in situ acetoxyborohydride reducible mixture significantly increases the size of the conjugates, resulting in a comparable sugar to carrier protein ratio (Ps:Pr) and a significantly lower % free polysaccharide.

[0098] Example 5: Acetoxy Hydrogenation Effect of temperature and holding time of boron-reducing mixture on conjugate size The effect of in situ acetoxyborohydride mixture preparation temperature on conjugate size was evaluated by preparing the reducing mixture at either 22°C or 40-50°C. Three different serotypes of Streptococcus pneumoniae (S. pneumoniae), ST-22F, ST-9N, and ST-35B, were used in this study. The results are shown in Figure 3. Conjugation was more effective for ST-9N and ST-35B when the reducing mixture was prepared at 22°C rather than 40-50°C, but there was no difference for ST-22F.

[0099] The size of the conjugates obtained using the in situ acetoxyborohydride reducing mixture depends on the reduction potential of the mixture. Of the three reducing species in the reducing mixture, SMAB has the highest reduction potential (Gribble GW, Chemical Society Reviews, 1998, Vol. 27, 395-404), potentially resulting in larger-than-desired conjugate sizes. Therefore, it is advisable to store the reducing mixture for a period of time after preparation and before use to reduce or completely deplete the amount of SMAB. However, over time, the amount of SDAB in the reducing mixture decreases, resulting in smaller conjugate sizes. The latter effect is demonstrated in Table 3 below using conjugates of bacterial capsular polysaccharides 9N, 22F, and 35B.

[0100] Table 3 [Table 3]

[0101] Example 6: In situ acetoxylation Hydrogenation Results of immunization of mice with pneumococcal conjugates prepared using a boron-reducing mixture Young female CD1 mice (6–8 weeks old, n = 10 / group) were immunized intramuscularly with 0.1 ml of 9N-CRM197, 22F-CRM197, or 35B-CRM197 vaccine on days 0 and 14. The 9N-CRM197, 22F-CRM197, or 35B-CRM197 vaccine was administered at 0.4 pg of 9N, 22F, or 35B polysaccharide conjugated to CRM197 per immunization. Serum was collected prior to the start of the study (pre-immunization) and on day 21 (post-dose 2, PD2). Mice were observed at least daily by trained animal care staff for signs of illness or distress. No vaccine-related adverse events were observed, and the vaccine formulation was deemed safe and well-tolerated in mice. All animal experiments were conducted in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mouse experimental protocols were approved by the Merck & Co., Inc. Institutional Animal Care and Use Committee.

[0102] Functional antibody levels were determined using an opsonophagocytosis assay (OPA) based on a protocol previously described at www.vaccine.uab.edu and software licensed by the University of Alabama (UAB) Research Foundation, Opsoiter®, and are briefly described below. Mouse sera were pooled for each group and tested using the OPA to determine functional antibody titers after two immunizations. ST-9N, ST-22F, and ST-35B conjugates (Arm 2) using the in situ acetoxyborohydride mixture described herein demonstrated higher functional antibody titers compared to the same conjugations without the in situ acetoxyborohydride mixture (Arm 1) (see Figures 4A, 4B, and 4C). In Arm 1, cyanoborohydride was used as the reducing agent for serotypes 9N and 22F, but no reducing agent was used for serotype 35B.

[0103] Incorporation by Reference The entire disclosure of each patent document and scientific article referenced herein is hereby incorporated by reference.

[0104] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The various structural elements of the different embodiments and the various disclosed method steps may be utilized in various combinations and permutations, and all such variations are to be considered aspects of the invention. The scope of the invention is, therefore, indicated by the appended claims, rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A method for conjugating an antigen to a carrier protein, comprising: a) activating an antigen to form an activated antigen; b) reacting the activated antigen with a carrier protein to obtain an intermediate, wherein the activated antigen and the carrier protein are conjugated via an imine group; and c) (i) combining acetic acid with a borohydride solution to form a reducing mixture comprising acetoxyborohydride; and (ii) treating said intermediate with said reducing mixture, wherein at least about 20% of the acetoxyborohydrides in said reducing mixture are diacetoxyborohydrides; reducing the imine group by a process comprising: thereby producing a conjugate antigen, wherein the antigen is a saccharide. method.

2. 2. The method of claim 1, wherein in step (ii), the remainder in the reducing mixture is either triacetoxyborohydride or a mixture of triacetoxyborohydride and monoacetoxyborohydride.

3. 3. The method of claim 1 or claim 2, wherein the saccharide is a bacterial capsular polysaccharide.

4. 4. The method of any one of claims 1 to 3, wherein the reducing mixture is held for a period of at least about 30 minutes before treating the intermediate with the reducing mixture.

5. 5. The method of claim 1, wherein the borohydride is sodium borohydride or potassium borohydride.

6. 6. The method of any one of claims 1 to 5, wherein the borohydride solution is prepared by dissolving the borohydride in dimethyl sulfoxide (DMSO).

7. 7. The method of any one of claims 1 to 6, wherein step (c)(i) is carried out at a temperature of from about 20°C to about 35°C.

8. 8. The method of claim 7, wherein step (c)(i) is carried out at a temperature of about 20°C to about 25°C.

9. 9. The method according to claim 1, wherein unreacted carbonyl groups are reduced by the reducing mixture.

10. 9. The method of any one of claims 1 to 8, further comprising reducing any unreacted carbonyl groups using borohydride.

11. 11. The method of any one of claims 1 to 10, wherein the reducing mixture is held for about 1 to about 8 hours before use.

12. 12. The method of claim 11, wherein the reducing mixture is held for about 2 to about 6 hours before use.

13. 13. The method of any one of claims 1 to 12, wherein the reducing mixture comprises at least about 25% diacetoxyborohydride.

14. The bacterial capsular polysaccharides are capable of inhibiting the production of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella typhimurium, Salmonella enterica, Salmonella typhimurium ...

6. The method according to any one of the preceding claims, wherein the antibacterial agent is derived from Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus aureus (Staphylococcus epidermidis), Staphylococcus erythrodermidis ...

15. 15. The method of claim 14, wherein the bacterial capsular polysaccharide is derived from Streptococcus pneumoniae (S. pneumoniae).

16. 16. The method of claim 15, wherein the S. pneumoniae capsular polysaccharide is of a serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, deOAc-15B, 16F, 17F, 18C, 19A, 19F, 20, 20A, 22F, 23A, 23F, 23B, 24F, 31, 33F, and 35B.

17. 15. The method of claim 14, wherein the bacterial capsular polysaccharide is from Neisseria meningitidis (N. meningitidis).

18. 18. The method of claim 17, wherein the N. meningitidis capsular polysaccharide is of a serotype selected from the group consisting of A, B, C, W135, X, and Y.

19. 19. The method of any one of claims 1 to 18, wherein the carrier protein is a protein selected from the group consisting of tetanus toxoid (TT), tetanus toxoid fragment C, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD (pneumococcal histidine triad protein D), PhtDE, and N19.

20. 20. The method of claim 19, wherein the carrier protein is CRM197.

21. 21. The method of claim 20, wherein the saccharide is conjugated to a lysine residue of CRM197 to yield a molar ratio of conjugated CRM197 lysine residues to total CRM197 amine residues of about 0.5:10 to about 5:

10.

22. 22. The method of any one of claims 1 to 21, wherein the conjugated antigen has a molecular weight of about 50 kDa to about 20,000 kDa.

23. 23. The method of any one of claims 1 to 22, wherein the conjugated antigen comprises less than about 45% free bacterial capsular polysaccharides compared to the total amount of bacterial capsular polysaccharides.

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