Conjugation of carbohydrate antigens using acetoxyborohydride

Abstract

The present disclosure generally relates to improved methods for preparing glycoconjugates. The method includes conjugating a saccharide to a carrier protein using an acetoxyborohydride-containing reducing mixture prepared in situ.

Description

Field of the Invention

[0001] The present invention relates to a method for preparing glycoconjugates by conjugating a polysaccharide to a carrier protein using in situ prepared acetoxyborohydride-mediated reductive amination. Background of the Invention

[0003] Bacterial capsular polysaccharides are long polymers composed of many repeating monosaccharide units that help protect bacteria from phagocytosis. Antibodies against the capsular polysaccharides of many pathogenic bacteria can enhance the phagocytosis of bacteria, thereby stimulating an immune response. However, although vaccines composed of purified polysaccharides are partially immunogenic in adults, they are unable to induce an antibody response in infants and young children. This problem is overcome by chemically conjugating the polysaccharide to a carrier protein, thereby making the polysaccharide more immunogenic. Conjugation converts the polysaccharide (a non-T cell-dependent antigen) into a protein (a T cell-dependent antigen), which enables isotype switching, affinity maturation, and the formation of memory B cells.

[0004] The two most widely used methods for conjugating bacterial polysaccharides to carrier proteins involve amidation and reductive amination. In the method involving amidation, the reducing end of the polysaccharide is first oxidized to the corresponding aldonic acid. This step is followed by the reaction of the acid group of the aldonic acid with the primary amine in the lysine side chain of the carrier protein. In the method involving reductive amination, the reducing end of the polysaccharide is first oxidized to the corresponding aldehyde. After this reaction, a Schiff base is formed between the aldehyde group and the primary amine (or N-terminal amine) in the lysine side chain of the carrier protein to generate an imine, and the imine is reduced with a hydride source. Cyanoborohydride has been used as the hydride source for the conjugation of bacterial polysaccharides (such as Streptococcus pneumoniae saccharides) (WO 1987 / 006838). However, the use of cyanoborohydride has disadvantages such as slow reaction time and contamination of the resulting amine with cyanide (Ahmed F. et al., J. Org. Chem., 1996, 61:3849-3862).

[0005] Triacetoxyborohydride has been used as an alternative to cyanoborohydride for 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). Triacetoxyborohydride has also been used for the reduction of imines formed during the conjugation of capsular polysaccharides to protein carriers (EP 2683408). However, there is a need to improve the efficiency of triacetoxyborohydride-mediated reductive amination. Summary of the Invention

[0007] Accordingly, the present invention provides an improved method for conjugating a carbohydrate antigen to a carrier protein using acetoxyborohydride. The improved method comprises reductive amination of the Schiff base formed between the carbohydrate and the carrier protein during the conjugation process using a reducing mixture comprising freshly prepared or in-situ prepared acetoxyborohydride.

[0008] In one aspect, the present disclosure provides a method for conjugating an antigen to a carrier protein. The method comprises the steps of: (a) activating the 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 linked by an imine group; and (c) reducing the imine group by a method comprising (i) mixing acetic acid with a borohydride solution to prepare a reducing mixture comprising acetoxyborohydride, and (ii) treating the intermediate with the reducing mixture. At least about 20% of the acetoxyborohydride in the reducing mixture used to treat the intermediate is diacetoxyborohydride (DAB). The antigen is a carbohydrate. The reducing mixture is alternatively referred to herein as an in-situ acetoxyborohydride reducing mixture.

[0009] In some embodiments, the remainder of the acetoxyborohydride in the reducing mixture is triacetoxyborohydride (TAB) or a mixture of TAB and monoacetoxyborohydride (MAB).

[0010] In some embodiments, the carbohydrate is a bacterial capsular polysaccharide.

[0011] In some embodiments, the reducing mixture is held for a period of time before being used to treat the intermediate (i.e., not used for a period of time after preparation of the reducing mixture), such as at least about 30 minutes, about 1 hour to about 8 hours, or about 2 hours to about 6 hours.

[0012] In some embodiments, the borohydride used is sodium borohydride or potassium borohydride. In some embodiments, the borohydride solution is prepared by dissolving the borohydride in dimethyl sulfoxide (DMSO).

[0013] 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.

[0014] In some embodiments, unreacted carbonyl groups (such as residual aldehyde groups) are reduced by the reducing mixture. In some embodiments, the method further comprises reducing unreacted carbonyl groups using a borohydride (such as sodium borohydride or potassium borohydride). In some embodiments, the reducing mixture comprises at least about 30% diacetoxyborohydride.

[0015] In some embodiments, the bacterial capsular saccharides conjugated according to the above methods are derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella vi, or Staphylococcus epidermidis.

[0016] In some embodiments, the carrier protein can be tetanus toxoid (TT), fragment C of tetanus toxoid, 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 the lysine residues of CRM197 such that the molar ratio of conjugated CRM197 lysine residues to total CRM197 amine residues is from about 0.5:10 to about 5:10.

[0017] In some embodiments, the conjugated antigen prepared according to the methods of the invention has a molecular weight of from about 50 kDa to about 20,000 kDa. In some embodiments, the conjugated antigen has less than about 45% free bacterial capsular polysaccharide, compared to the total amount of bacterial capsular polysaccharide.

[0018] In another aspect, the present disclosure provides an immunogenic composition comprising a conjugated antigen or a mixture of conjugated antigens admixed with a pharmaceutically acceptable excipient, wherein one or more of the conjugated antigens are prepared according to the conjugation methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a graph showing the concentration changes over time of sodium monoacetoxyborohydride (SMAB), sodium diacetoxyborohydride (SDAB), and sodium triacetoxyborohydride (STAB) in an in situ acetoxyborohydride reducing mixture. Figure 1 Also shown is the relative amount of SDAB and STAB present in a commercially available STAB.

[0021] Figure 2The size of the conjugate formed is shown as a function of the holding time of the in situ acetoxyborohydride reducing mixture before it is used to reduce the imine group in the conjugation reaction.

[0022] Figure 3 The effect of temperature during preparation of the in situ acetoxyborohydride reducing mixture on conjugation efficiency is shown. Conjugation efficiency was measured by the size of the conjugate produced (n=3).

[0023] Figure 4( Figure 4A 、 Figure 4B and Figure 4C ) is a graph showing functional antibody titers (OPA titers) in mice immunized with pneumococcal serotypes 9N, 22F, and 35B. In the conjugation of these serotypes, an in situ acetoxyborohydride reducing mixture was used (Group 2) or not (Group 1). In Group 1, cyanoborohydride was used as a reducing agent for serotypes 9N and 22F, while no reducing agent was used for serotype 35B. Detailed Description of the Invention

[0025] The present disclosure may be more readily understood by reference to the following detailed description of various embodiments of the present disclosure and the examples included herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In the description of the embodiments and in the claims, certain terms will be used according to the definitions given below.

[0026] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein and / or which will become apparent to one of ordinary skill in the art upon reading this disclosure.

[0027] As used herein, the term "approximately" refers to a statistically significant range of a numerical value, such as a specified concentration range, time range, molecular weight, temperature, or pH. Such a range may be on the order of typically 10%, more typically 5%, or 1% of a given numerical value or range. Sometimes, such a range may be within the typical experimental error of a standard method for measuring and / or determining a given value or range. The tolerances encompassed by the term "approximately" depend on the specific system under investigation and are readily understood by one of ordinary skill in the art. Whenever a range is listed in this application, each endpoint of the range and each value within the range are also considered embodiments of the present disclosure.

[0028] It is noted that in the present disclosure, terms such as “comprises” and “comprising” mean “including” etc. Such terms mean that a specific component or a group of components are included without excluding any other components.

[0029] Terms such as "consisting essentially of" allow for additional components or steps that do not detract from the novel or essential features of the disclosure, i.e., they exclude additional unrecited components or steps that would detract from the novel or essential features of the disclosure. The term "consisting of" is a closed term. Thus, these terms are meant to include the specified components or group of components and to exclude all other components.

[0030] As used herein, the term "saccharide" is used to refer to polysaccharides, oligosaccharides, or monosaccharides.

[0031] As used herein, the term "conjugate" or "glycoconjugate" refers to a saccharide covalently conjugated to a carrier protein. The glycoconjugates of the present disclosure and immunogenic compositions containing them may contain a certain amount of free saccharides.

[0032] As used herein, the term "free saccharide" refers to a saccharide that is not covalently conjugated to a carrier protein but is still present in the glycoconjugate composition. Free saccharides may be non-covalently associated (i.e., non-covalently bound, adsorbed, or entrapped within or together with) with the conjugated sugar-carrier protein glycoconjugate. The terms "free polysaccharide" and "free capsular polysaccharide" are used herein to express the same meaning with respect to glycoconjugates, where the saccharide is a polysaccharide or a capsular polysaccharide, respectively.

[0033] As used herein, "freshly or in situ prepared acetylborohydride" refers to a mixture of acetylborohydrides prepared by mixing borohydride (e.g., sodium borohydride) and acetic acid less than 25 hours prior to use in a conjugation reaction, which contains diacetylborohydride, triacetylborohydride, and optionally monoacetylborohydride. For example, acetylborohydride can be prepared 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or 30 minutes prior to use.

[0034] As used herein, "conjugation" refers to the process by which a saccharide (e.g., a bacterial capsular polysaccharide) is covalently linked to a carrier molecule or carrier protein. Conjugation can be carried out according to the methods described below or by other methods known in the art. Conjugation enhances the immunogenicity of the bacterial capsular polysaccharide.

[0035] The term "subject" refers to a mammal, including a human, or to a bird, fish, reptile, amphibian, or any other animal. The term "subject" also includes domestic pets or 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 livestock animals. Non-limiting examples of livestock animals include alpacas, bison, camels, cows, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

[0036] Glycoconjugate

[0037] The present disclosure relates to methods of conjugating saccharides to carrier proteins, i.e., methods of preparing glycoconjugates, particularly by reducing imine groups formed during conjugation using a reducing mixture comprising diacetoxyborohydride, triacetoxyborohydride, and optionally monoacetoxyborohydride, which is freshly prepared or prepared in situ.

[0038] In the glycoconjugates of the present disclosure, the saccharide can be a monosaccharide, oligosaccharide, or polysaccharide, and the carrier protein can be any suitable carrier protein as further described herein or known to those of skill in the art. In some embodiments, the saccharide is a polysaccharide, particularly a bacterial capsular polysaccharide, such as a polysaccharide from Streptococcus pneumoniae (S. pneumoniae; serotype 35B). In some embodiments, the carrier protein is CRM 197 (cross-reactive material 197 from Corynebacterium diphtheriae C7).

[0039] Capsular polysaccharides can be prepared by standard techniques known to those of skill in the art and 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. The conjugates can be prepared by separate methods and formulated into single-dose preparations. For example, each pneumococcal polysaccharide serotype can be grown separately, and then each polysaccharide can be purified by steps including one or more of centrifugation, precipitation, ultrafiltration, and column chromatography. The purified polysaccharide can be chemically activated to enable the saccharide to react with the carrier protein. Once activated, each capsular polysaccharide can be conjugated separately to the carrier protein to form a glycoconjugate. Each capsular polysaccharide in the preparation can be conjugated to the same carrier protein. Alternatively, more than one carrier protein can be used to conjugate the polysaccharide. Chemical activation of the polysaccharide can be achieved by conventional means. See, e.g., U.S. Patent Nos. 4,902,506, 7,709,001, and 7,955,605.

[0040] In one embodiment, the glycoconjugate of the present disclosure has a molecular weight of from about 50 kDa to about 20,000 kDa. In another embodiment, the glycoconjugate has a molecular weight of from about 200 kDa to about 10,000 kDa. In another embodiment, the glycoconjugate has a molecular weight of from about 500 kDa to about 5,000 kDa. In one embodiment, the glycoconjugate has a molecular weight of from about 1,000 kDa to about 3,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of from about 600 kDa to about 2800 kDa; from about 700 kDa to about 2700 kDa; from about 1000 kDa to about 2000 kDa; from about 1800 kDa to about 2500 kDa; from about 1100 kDa to about 2200 kDa; from about 1900 kDa to about 2700 kDa; from about 1200 kDa to about 2400 kDa; from about 1700 kDa to about 2600 kDa; from about 1300 kDa to about 2600 kDa; from about 1600 kDa to about 3000 kDa. Any integer within any of the above ranges is considered an embodiment of the present disclosure.

[0041] In one embodiment, the polysaccharide is a capsular polysaccharide derived from Neisseria meningitidis (N. meningitidis). In some such embodiments, the capsular polysaccharide is selected from the serotype A, B, C, W135, X, and Y capsular polysaccharides of Neisseria meningitidis.

[0042] In one embodiment, the polysaccharide is a capsular polysaccharide derived 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.

[0043] 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, the de-O-acetylated serotype 15B pneumococcal polysaccharide and the serotype 15C pneumococcal polysaccharide can each have an O-acetyl content per repeating unit in the range of 0 - 5%, or in the range of 0 - 4%, or in the range of 0 - 3%, or in the range of 0 - 2%, or in the range of 0 - 1%, or in the range of 0 - 0.5%, or in the range of 0 - 0.1%, or no O-acetyl content. In the report of Spencer B.L. et al., pneumococcal polysaccharide 15C can be slightly O-acetylated (Spencer, B.L. et al., Clin. Vac. Immuno. (2017) 24(8):1 - 13). Thus, in any embodiment of the methods described herein, de-O-acetylated serotype 15B (DeOAc15B) can be used in place of serotype 15C. Methods for de-O-acetylation are known in the art, for example, as described in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9):1223 - 1227.

[0044] In some embodiments, the glycoconjugates of the present disclosure comprise a bacterial capsular polysaccharide, wherein the capsular polysaccharide has a molecular weight of 10 kDa to 2,000 kDa or 50 kDa to 1,000 kDa.

[0045] The glycoconjugates comprising a capsular polysaccharide covalently conjugated to a carrier protein produced by the methods of the present invention can have one or more of the following characteristics: the molecular weight of the polysaccharide is 50 kDa to 1,000 kDa; the molecular weight of the glycoconjugate is 1,000 kDa to 5,000 Kda; the conjugate comprises less than about 45% free polysaccharide relative to the total polysaccharide. In some embodiments, the molecular weight of the polysaccharide is 10 kDa to 2,000 kDa. In some embodiments, the molecular weight of the glycoconjugate is 50 kDa to 20,000 kDa. In other embodiments, the molecular weight of the glycoconjugate is 200 kDa to 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 over time, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 120 days or even longer after preparation of the conjugate.

[0046] 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 and the N-terminal amine (i.e., a total of 40 amine residues) can serve as conjugation sites. However, not all of these amine residues have equal reactivity. In some embodiments, in a glycoconjugate made with CRM197, only 4 to 15 lysine residues are covalently linked to the saccharide, such that the molar ratio of conjugated lysine residues to total CRM197 amine residues is from about 1:10 to about 4:10. In another embodiment, 2 to 20 lysine residues of CRM197 are covalently linked to the saccharide, such that the molar ratio of conjugated lysine residues to CRM197 amine residues is from about 0.5:10 to about 5:10.

[0047] The saccharide:carrier protein ratio (w / w) can also vary. In one embodiment, the saccharide:carrier protein ratio (w / w) is from 0.2 to 4. In another embodiment, the saccharide:carrier protein ratio (w / w) is from 1.1 to 1.7. In some embodiments, the saccharide is a bacterial capsular polysaccharide, and the saccharide:carrier protein ratio (w / w) is from 0.2 to 4. In other embodiments, the saccharide is a bacterial capsular polysaccharide, and the saccharide:carrier protein ratio (w / w) is from 1.1 to 1.7. In some such embodiments, the carrier protein is CRM197.

[0048] The frequency of linkage of the sugar chain to lysine residues on the carrier protein can also vary. For example, in one embodiment, for every 100 sugar repeat units of the polysaccharide, there is at least one covalent linkage between the carrier protein and the polysaccharide. In one embodiment, for every 50 sugar repeat units of the polysaccharide, there is at least one covalent linkage between the carrier protein and the polysaccharide. In one embodiment, for every 25 sugar repeat units of the polysaccharide, there is at least one covalent linkage between the carrier protein and the polysaccharide. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 4 sugar repeat units of the polysaccharide. In another embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 10 sugar repeat units of the polysaccharide. In a further embodiment, a covalent linkage between the carrier protein and the polysaccharide occurs at least once in every 15 sugar repeat units of the polysaccharide. In another embodiment, 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 sugar repeat units of the polysaccharide, there is at least one linkage between the carrier protein and the sugar.

[0049] In some embodiments, the carrier protein is CRM197, and the covalent linkage between CRM197 and the polysaccharide occurs at least once per 4, 10, 15, or 25 sugar repeat units of the polysaccharide. In some such embodiments, the polysaccharide is a bacterial capsular polysaccharide, such as a capsular polysaccharide derived from Streptococcus pneumoniae (S. pneumoniae) or Neisseria meningitidis (N. meningitidis).

[0050] In one embodiment, the glycoconjugate comprises less than about 45% free carbohydrate, compared to the total amount of carbohydrate. In another embodiment, the glycoconjugate comprises less than about 30% free carbohydrate, compared to the total amount of carbohydrate. In another embodiment, the glycoconjugate comprises less than about 20% free carbohydrate, compared to the total amount of carbohydrate. In a further embodiment, the glycoconjugate comprises less than about 10% free carbohydrate, compared to the total amount of carbohydrate. In another embodiment, the glycoconjugate comprises less than about 5% free carbohydrate, compared to the total amount of carbohydrate.

[0051] In some embodiments, the glycoconjugate comprises 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, compared to the total amount of the glycoconjugate.

[0052] Carrier protein

[0053] The term "protein carrier" or "carrier protein" or "carrier" refers to any protein molecule that can be conjugated to an antigen, such as a capsular polysaccharide, for which an immune response is desired.

[0054] Conjugation to a carrier can enhance the immunogenicity of an antigen. Protein carriers for antigens can be toxins, toxoids, or any mutant cross-reactive material (CRM) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, Escherichia coli, Staphylococcus, and Streptococcus. In one embodiment, the carrier is diphtheria toxoid CRM.sub.197, which is derived from Corynebacterium diphtheriae strain C7(6197) that produces the CRM.sub.197 protein. The ATCC accession number of this strain is No. 53281. The method for producing CRM.sub.197 is described in U.S. Patent No. 5,614,382, which is hereby incorporated by reference in its entirety. Alternatively, fragments or epitopes of protein carriers or other immunogenic proteins can be used. For example, hapten-type antigens can be conjugated to T cell epitopes of bacterial toxins, toxoids, or CRMs. 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 LT, Escherichia coli ST, and exotoxin A from Pseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin (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 (PPD) of tuberculin can also be used as carrier proteins.

[0055] Characterization of polysaccharide-protein conjugates

[0056] Glycoconjugates can be characterized based on their molecular weight, which can be the number-average molecular weight (Mn) or the weight-average molecular weight (Mw). Glycoconjugates are polymers that exist with a certain distribution of chain lengths and molecular weights. Therefore, it is not possible to have an exact molecular weight for a glycoconjugate. Instead, the average values of different parameters are used to indicate the molecular weight of the glycoconjugate. Mn and Mw are two such average values. Mn is the statistical average molecular weight of all the polymer chains in a sample. It is obtained 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 chains. It is based on the fact that molecules with larger masses contribute a greater share to the total mass of the polymer sample than smaller molecules. Therefore, Mw is a weighted average and reflects the average weight of the molecules in the polymer sample. Different methods are used to determine Mn and Mw. For glycoconjugates, size-exclusion chromatography can be used to measure Mn, and static light scattering can be used to measure Mw. Mn and Mw can be measured simultaneously using size-exclusion chromatography (SEC) followed by continuous monitoring of differential refractive index (RI), ultraviolet light absorbance (UV), and multi-angle laser light scattering (MALS). Pollock JF et al., Bioconjug Chem. September 19, 2012; 23(9):1794–1801.

[0057] Glycoconjugates can also be characterized by the number of lysine residues conjugated to carbohydrates in the carrier protein. This value can be expressed as a range of conjugated lysine residues (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent attachment to the polysaccharide can be obtained by amino acid analysis using conventional methods known to those of ordinary skill in the art. Conjugation results in a decrease in the number of recovered lysine residues compared to the carrier protein starting material used to generate the conjugate material. For example, the degree of conjugation can be as low as 2 or as high as 15.

[0058] Another method of characterizing glycoconjugates is by using the ratio of carbohydrate to carrier protein (weight / weight) (Ps:Pr). For example, Ps:Pr can be as low as 0.5 or as high as 3.0. Glycoconjugates can contain free carbohydrates that are not covalently conjugated to the carrier protein but are still present in the glycoconjugate composition. The free carbohydrates can be non-covalently associated with the glycoconjugate (i.e., non-covalently bound, adsorbed, or entrapped within or together with it). For example, compared to the total amount of polysaccharide, the glycoconjugate can contain less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide.

[0059] Conjugation

[0060] To prepare conjugates with carrier proteins, polysaccharides must be activated (i.e., chemically modified) before they can be chemically linked to a carrier such as a protein. Prior to the activation step, the saccharides can be hydrolyzed or mechanically fractionated by pressure homogenization to achieve an appropriate molecular weight for activation and subsequent conjugation (e.g., 50 kDa to 500 kDa). Partial oxidation of the carbohydrates in the polysaccharide has been effectively utilized to generate aldehyde groups, which are then coupled to amino groups such as the lysine residues of the carrier protein to generate immunogenic conjugates. Importantly, the methods used to conjugate polysaccharides to carrier proteins result in stable covalent linkages and the reaction conditions are mild enough to maintain the structural integrity of the individual components. Methods commonly used to activate polysaccharides and conjugate them to carrier proteins include reductive amination chemistry (RAC), cyanation, and the use of carbodiimides. Reductive amination generally involves the selective oxidation of vicinal -OH groups to reactive aldehyde groups using sodium periodate or potassium periodate or periodic acid. Cyanation is used to randomly convert -OH groups to reactive -CN groups. Carbodiimides are used to activate carboxyl groups by replacing -OH groups with carbodiimides.

[0061] Reductive amination chemistry (RAC) is one of the most commonly used methods for conjugating polysaccharides to proteins because 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 is typically selectively reduced to a stable saturated carbon-nitrogen bond in the presence of sodium cyanoborohydride. In addition, reductive amination can be carried out in aqueous solution under mild conditions sufficient to maintain the structural integrity of the saccharide and protein components. After conjugation, the unreacted aldehyde can then be reduced by sodium borohydride. The conjugate can then be purified, for example, by ultrafiltration / diafiltration.

[0062] However, as described in EP 2683408B1, the use of cyanoborohydride ions has certain disadvantages, including relatively slow reaction times and the possibility of contamination of the conjugate with cyanide (Ahmed F. et al., J. Org. Chem., 1996, 61:3849-3862). The use of microwave radiation has been proposed as a solution to the slow reaction times (EP 1035137). EP2683408B1 proposes the use of triacetoxyborohydride (BH(OA)3) as an alternative to cyanoborohydride because reductive amination using the triacetoxyborohydride anion is faster than the equivalent reaction using cyanoborohydride ions and does not produce toxic by-products. The proposal to use triacetoxyborohydride in the conjugation of bacterial capsular polysaccharides is based on its previously reported use in the reductive amination of aldehydes and ketones (J. Org. Chem., 1996, 61:3849-3862) as well as carbohydrates (Dalpathado et al., Anal. Bioanal. Chem. (2005) 281:130-1137).

[0063] However, the present inventors have found that a reductive amination method using triacetoxyborohydride obtained from commercial sources is not very effective and have found an improvement to this method. This improvement involves using a freshly prepared reductive mixture containing acetoxyborohydride for reductive amination. It has been observed that a reductive mixture prepared by mixing sodium borohydride and acetic acid contains a higher concentration of DAB (diacetoxyborohydride) when fresh - within about 15 hours of preparation - than commercially obtained triacetoxyborohydride reagent. See Figure 1 . It has also been observed that over time, the concentration of DAB in this reductive mixture decreases ( Figure 1 ), and this decrease parallels a decrease in the size of the conjugate produced. See Figure 2 . In other words, a reductive mixture containing a higher concentration of DAB results in a larger amount of larger-sized conjugates. It should be noted that among MAB, DAB, and TAB, MAB has the highest reduction potential, DAB the second highest, and TAB the lowest reduction potential. Since the in-situ prepared acetoxyborohydride reductive mixture has a relatively higher proportion of acetoxyborohydride species with a higher reduction potential compared to commercially available TAB, improved conjugation and conjugation properties are observed when using the in-situ prepared acetoxyborohydride reductive mixture (Gordon W. Gribble. Chem. Soc. Rev., 1998, 27: 395 - 404).

[0064] It has also been observed that holding the reaction mixture for a period of time, such as 1 hour, before using it in the conjugation reaction results in consistency in the size of the conjugate and thus in the production. The various aspects of conjugating carbohydrate antigens using the freshly prepared acetoxyborohydride reductive mixture described herein are elaborated in more detail in the numbered embodiments below.

[0065] Embodiment 1 provides a method for conjugating an antigen to a carrier protein, the method comprising the steps of:

[0066] a) activating the antigen to form an activated antigen;

[0067] b) reacting the activated antigen with the carrier protein to obtain an intermediate, wherein the activated antigen and the carrier protein are linked by an imine group; and

[0068] c) reducing the imine group by a method comprising

[0069] (i) mixing acetic acid with a borohydride solution to prepare a reductive mixture containing acetoxyborohydride, and

[0070] (ii) treating the intermediate with the reducing mixture, wherein at least about 20% of the acetoxyborohydride in the reducing mixture is diacetoxyborohydride;

[0071] Thereby producing a conjugated antigen, wherein the antigen is a saccharide.

[0072] Embodiment 2 provides the method of Embodiment 1, wherein in step (ii), the remainder of the acetoxyborohydride in the reducing mixture is triacetoxyborohydride or a mixture of triacetoxyborohydride and monoacetoxyborohydride.

[0073] Embodiment 3 provides the method of Embodiment 1 or 2, wherein the saccharide is a bacterial capsular polysaccharide.

[0074] Embodiment 4 provides the method of any one of Embodiments 1-3, wherein the reducing mixture is maintained for at least 30 minutes before treating the intermediate with the reducing mixture.

[0075] Embodiment 5 provides the method of any one of Embodiments 1-4, wherein the borohydride is sodium borohydride or potassium borohydride.

[0076] Embodiment 6 provides the method of any one 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 solvents known to those of ordinary skill in the art can be used.

[0077] Embodiment 7 provides the method of any one of Embodiments 1-6, wherein step (c)(i) is carried out at a temperature of about 20 °C to about 35 °C.

[0078] 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.

[0079] Embodiment 9 provides the method of any one of Embodiments 1-8, wherein unreacted carbonyl groups (such as residual aldehyde groups) are reduced by the reducing mixture.

[0080] Embodiment 10 provides the method of any one of Embodiments 1-8, which further comprises reducing unreacted carbonyl groups using a borohydride such as sodium borohydride or potassium borohydride.

[0081] Embodiment 11 provides the method of any one of Embodiments 1-10, wherein the reducing mixture is maintained for about 1 to about 8 hours before use.

[0082] Embodiment 12 provides the method of Embodiment 11, wherein the reducing mixture is maintained for about 2 to about 6 hours before use.

[0083] Embodiment 13 provides the method of any one of Embodiments 1-12, wherein the reducing mixture comprises at least about 25% of diacetoxyborohydride or at least about 30% of diacetoxyborohydride.

[0084] Embodiment 14 provides the method of any one of Embodiments 1-13, wherein the bacterial capsular saccharides are derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella vi or Staphylococcus epidermidis.

[0085] Embodiment 15 provides the method of Embodiment 14, wherein the bacterial capsular polysaccharide is derived from Streptococcus pneumoniae (S. pneumoniae).

[0086] Embodiment 16 provides the method of Embodiment 15, wherein the Streptococcus pneumoniae capsular polysaccharide has a serotype selected from 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.

[0087] Embodiment 17 provides the method of Embodiment 14, wherein the bacterial capsular saccharides are derived from Neisseria meningitidis (N. meningitidis).

[0088] Embodiment 18 provides the method of Embodiment 17, wherein the Neisseria meningitidis capsular polysaccharide has a serotype selected from A, B, C, W135, X and Y.

[0089] Embodiment 19 provides the method of any one of Embodiments 1-18, wherein the carrier protein is a protein selected from tetanus toxoid (TT), fragment C of tetanus toxoid, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD (pneumococcal histidine triad protein D), PhtDE, and N19.

[0090] Embodiment 20 provides the method of Embodiment 19, wherein the carrier protein is CRM197.

[0091] Embodiment 21 provides the method of Embodiment 20, wherein the saccharide is conjugated to a lysine residue of CRM197 such that the molar ratio of the conjugated CRM197 lysine residue to the total CRM197 amine residues is from about 0.5:10 to about 5:10.

[0092] Embodiment 22 provides the method of any one of Embodiments 1-21, wherein the conjugated antigen has a molecular weight of from about 50 kDa to about 20,000 kDa.

[0093] Embodiment 23 provides the method of any one of Embodiments 1-22, wherein the conjugated antigen comprises less than about 45% free bacterial capsular polysaccharide compared to the total amount of the bacterial capsular polysaccharide saccharide.

[0094] Pharmaceutical composition

[0095] In another aspect, the present disclosure provides an immunogenic composition comprising the glycoconjugate of the present disclosure and at least one of an adjuvant, a diluent, or a carrier.

[0096] In some embodiments, the present disclosure provides an immunogenic composition comprising the 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 derived from Streptococcus pneumoniae (S. pneumoniae) or Neisseria meningitidis (N. meningitidis).

[0097] In some embodiments, the immunogenic composition comprises an adjuvant. In some such embodiments, the adjuvant is an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate, and aluminum hydroxide. In one embodiment, the immunogenic composition comprises the adjuvant aluminum phosphate.

[0098] In some embodiments, the immunogenic composition comprises a conjugated antigen or a mixture of conjugated antigens admixed with a pharmaceutically acceptable excipient, wherein one or more of the conjugated antigens are prepared by the method of any one of the numbered Embodiments 1-23 above.

[0099] In some embodiments, the glycoconjugates or immunogenic compositions of the present disclosure can be used to generate functional antibodies as measured by killing bacteria in an animal efficacy model or by opsonophagocytic killing assays.

[0100] 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 the immunogenic composition of the present disclosure as described herein. In related embodiments, the present disclosure provides a method of inducing an immune response against a pathogenic bacterium in a subject, the method comprising administering to the subject an immunologically effective amount of the immunogenic composition as described herein.

[0101] In another aspect, the present disclosure provides a method of preventing or ameliorating a disease or condition in a subject caused by a pathogenic bacterium, the method comprising administering to the subject an immunologically effective amount of the immunogenic composition as described herein.

[0102] In another aspect, the present disclosure provides a method of reducing the severity of at least one symptom of a disease or condition in a subject caused by infection with a pathogenic bacterium, the method comprising administering to the subject an immunologically effective amount of the immunogenic composition as described herein.

[0103] In some embodiments, the pathogenic bacterium is Streptococcus pneumoniae or Neisseria meningitidis. In one embodiment, the pathogenic bacterium is Streptococcus pneumoniae and the capsular polysaccharide has a serotype selected from 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 Neisseria meningitidis and the capsular polysaccharide is selected from the serotype A, B, C, W-135, X and Y capsular polysaccharides of Neisseria meningitidis.

[0104] The invention will be more clearly understood from the following examples, which are given to illustrate the invention but not to limit its scope. Examples

[0105] Example 1: Preparation of an in situ acetoxyborohydride reducing mixture

[0106] An in-situ acetoxyborohydride reducing mixture is prepared by adding 0.4 M to 0.8 M of sodium borohydride to DMSO and mixing until the sodium borohydride is completely dissolved. Subsequently, approximately 3 to 5 molar equivalents of acetic acid are added to the sodium borohydride solution to form the in-situ acetoxyborohydride reducing mixture. This reducing mixture contains three reducing species, namely MAB, DAB, and TAB. These species have different reduction potentials. Additionally, the concentration of each species in the mixture varies with time and temperature.

[0107] The reaction for preparing the acetoxyborohydride reducing mixture is exothermic. The temperature increase during the preparation of the reducing mixture is restricted by adding acetic acid in multiple steps (4 to 8 additions). This preparation is carried out in a glove box to prevent the introduction of moisture. Moisture induces the hydrolysis of the reducing species and further raises the reaction temperature. After the preparation is completed, the reducing mixture is held (i.e., left aside) for approximately 2 - 8 hours before it is used in the conjugation reaction. It has been observed that in each preparation of the reducing mixture, by holding the mixture for the same amount of time before use, a roughly the same DAB concentration can be achieved in the mixture.

[0108] Example 2: Composition of an in situ acetoxyborohydride reducing mixture

[0109] 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 with time. These amounts can be measured by NMR. In this example, the measurement of the composition of the reducing mixture starts after the last addition of acetic acid to the sodium borohydride solution. MAB is the most unstable species in this reducing mixture. It is consumed within the first two hours of the reaction (see Figure 1 ). The DAB concentration in this reducing mixture decreases with time, but the rate of decrease slows down. This indicates that the DAB concentration eventually reaches a plateau. On the other hand, the concentration of TAB increases with time as MAB and DAB are converted into TAB.

[0110] Example 3: Between conjugate size and DAB concentration in an in situ acetoxyborohydride reducing mixture Correlation

[0111] A study was conducted to examine whether there is a correlation between the DAB concentration in the in-situ acetoxyborohydride reducing mixture and the conjugate size. The conjugate size reflects the efficiency of the conjugation method. The larger the conjugate size, the more efficient the method. In one study, using the bacterial capsular polysaccharide ST-35B as an antigen, it was observed that over time, as the DAB concentration in the reducing mixture decreased, the size of the ST-35B conjugate also decreased (see Figure 2)。Observation was carried out over 25 hours. The fact that the amount of TAB increased while the amount of DAB decreased in the reducing mixture during this period strongly suggests that most of the reductive amination occurring during conjugate formation is attributed to reduction by DAB rather than TAB. In other words, DAB, rather than TAB, is the main reducing species. Note that the level of MAB reached zero within approximately 1 - 2 hours of preparing the reducing mixture.

[0112] Conjugation of several Streptococcus pneumoniae serotypes was carried out using an in - situ acetoxyborohydride reducing mixture. These Streptococcus pneumoniae serotypes and the conjugation results are shown in Table 1 below.

[0113] Table 1

[0114]

[0115]

[0116] Example 4: Comparison of an in situ acetoxyborohydride reducing mixture with commercial STAB

[0117] The concentrations of MAB, DAB, and TAB in the in - situ DAB reducing mixture were compared with the concentrations of these species in a commercial STAB product. As Figure 1 shown, in one of the commercial TABs tested, DAB and TAB were present at approximately 17% and 83% respectively. The commercial TAB product did not contain any MAB, which was as expected since MAB has low stability and is converted to TAB within approximately 1 - 2 hours.

[0118] Considering that DAB is the main reducing species (see Example 3) and considering that the level of DAB in the in - situ acetoxyborohydride reducing mixture is higher than in commercial STAB (at least during the 25 - hour period after preparation; see Figure 1 ), the in - situ acetoxyborohydride reducing mixture is a better reducing agent than commercial TAB. This is confirmed by a direct comparison of the conjugate sizes obtained using the in - situ acetoxyborohydride reducing mixture described herein with the conjugate sizes obtained using commercial STAB (see Table 2). The commercial and in - situ acetoxyborohydride were compared using the same molar equivalents. The results obtained using ST - 35B, ST - 9N, and ST - 22F as bacterial polysaccharides are shown in Table 2 below.

[0119] Table 2

[0120]

[0121]

[0122] Results showed that the in-situ acetoxyborohydride reducing mixture led to much higher conjugate sizes, comparable saccharide / carrier protein ratios (Ps:Pr), and much lower free polysaccharide %.

[0123] Example 5: Effect of temperature and holding time of an in situ acetoxyborohydride reducing mixture on conjugate size Effect

[0124] The effect of the preparation temperature of the in-situ acetoxyborohydride mixture on conjugate size was evaluated by preparing the reducing mixture at 22 °C or at 40 - 50 °C. Three different serotypes of Streptococcus pneumoniae, ST-22F, ST-9N, and ST-35B, were used in this study. Results showed that Figure 3 Conjugation of ST-9N and ST-35B was more efficient when the reducing mixture was prepared at 22 °C rather than at 40 - 50 °C, while in the case of ST-22F, there was no difference.

[0125] The size of the conjugates obtained using the in-situ acetoxyborohydride reducing mixture depends on the reduction potential of the mixture. Among the three reductants in the reducing mixture, SMAB has the highest reduction potential (Gribble GW, Chemical Society Reviews, 1998, Vol. 27, pp. 395 - 404) and can lead to conjugate sizes larger than the desired size. Therefore, it is advisable to keep the reducing mixture for some time after preparation 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 a decrease in conjugate size. The latter effect is shown in Table 3 below for the conjugation of bacterial capsular polysaccharides 9N, 22F, and 35B.

[0126] Table 3

[0127]

[0128] Example 6: Results of immunizing mice with a pneumococcal conjugate prepared using an in situ acetoxyborohydride reducing mixture Results

[0129] Female juvenile 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 day 0 and day 14. The 9N-CRM197, 22F-CRM197, or 35B-CRM197 vaccine was administered at 0.4 μg per immunization of 9N, 22F, or 35B polysaccharide conjugated to CRM197. Serum was collected before the start of the study (pre-immunization) and on day 21 (after dose 2, PD2). Trained animal care personnel observed the mice for any signs of disease or distress at least daily. The vaccine preparations were considered safe and well tolerated in the mice as no vaccine-related adverse events were noted. All animal experiments were conducted strictly in accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The mouse experimental protocol was approved by the Institutional Animal Care and Use Committee of Merck & Co., Inc.

[0130] Based on the foregoing protocol from www.vaccine.uab.edu and software owned and licensed by the University of Alabama (UAB) Research Foundation Functional antibody levels were determined using opsonophagocytic assays (OPA), which are briefly described below. The sera of the mice in each group were pooled and tested using OPA to determine the functional antibody titers after two immunizations. The ST-9N, ST-22F, and ST-35B conjugates (Group 2) using the in situ acetoxyborohydride mixture described herein showed higher functional antibody titers compared to the same conjugates (Group 1) without using the in situ acetoxyborohydride mixture (see Figure 4A 、 Figure 4B and Figure 4C ). In Group 1, cyanoborohydride was used as the reducing agent for serotypes 9N and 22F, while for serotype 35B, no reducing agent was used.

[0131] Incorporated herein by reference

[0132] The entire disclosure of each patent document and scientific article mentioned herein is incorporated herein by reference for all purposes.

[0133] Equivalents

[0134] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are, therefore, to be considered in all respects as illustrative and not restrictive of the invention described herein. The various structural elements of the different embodiments and the various disclosed method steps may be used in various combinations and permutations, and all such variations are considered to be forms of the present invention. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that fall within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

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

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

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

4. The method according to any one of claims 1-3, wherein the reducing mixture is maintained for at least 30 minutes before treating the intermediate with the reducing mixture.

5. The method according to any one of claims 1-4, wherein the borohydride is sodium borohydride or potassium borohydride.

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

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

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

9. The method according to any one of claims 1-8, wherein unreacted carbonyl groups are reduced by the reducing mixture.

10. The method according to any one of claims 1-8, which further comprises using borohydride to reduce unreacted carbonyl groups.

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

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

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

14. The method according to any one of the preceding claims, wherein the bacterial capsular saccharide is derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Salmonella vi or Staphylococcus epidermidis.

15. The method according to claim 14, wherein the bacterial capsular polysaccharide is derived from S. pneumoniae.

16. The method according to claim 15, wherein the S. pneumoniae capsular polysaccharide has a serotype selected from 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. The method according to claim 14, wherein the bacterial capsular saccharide is derived from N. meningitidis.

18. The method according to claim 17, wherein the N. meningitidis capsular polysaccharide has a serotype selected from A, B, C, W135, X and Y.

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

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

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

10.

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

23. The method according to any one of claims 1-22, wherein the conjugated antigen contains less than about 45% free bacterial capsular polysaccharide, compared to the total amount of the bacterial capsular saccharide.

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