Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Abstract

The present invention relates to novel conjugated capsular saccharide antigens (glycoconjugates), immunogenic compositions comprising said glycoconjugates, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to novel conjugated capsular saccharide antigens (glycoconjugates), immunogenic compositions comprising said glycoconjugates, and uses thereof. The immunogenic compositions of the invention typically comprise glycoconjugates, where the saccharide is derived from a bacterial capsular polysaccharide antigen, particularly a capsular polysaccharide derived from a pathogenic bacterium. The invention also relates to the vaccination of human subjects, particularly infants and the elderly, against infectious diseases using said glycoconjugates. [Background technology]

[0002] Both Gram-positive and Gram-negative bacteria can produce capsules, extracellular compartments that surround bacterial cells and often prevent reactions between internal cell surface antigens and their cognate antigens. Capsules have been found in several medically important bacteria, particularly extraintestinal and invasive strains. Bacterial capsules are composed mostly of polysaccharides, which form a gelatinous mass around the cells. Capsular polysaccharides are important immunogens, and they have become important components in vaccine design. They have proven useful in eliciting immune responses, especially when linked to carrier proteins.

[0003] The technique of increasing the immunogenicity of poorly immunogenic molecules by conjugating them to "carrier" molecules has been used successfully for decades (see, e.g., Goebel et al. (1939) J. Exp. Med. 69:53). For example, many immunogenic compositions have been described that exploit this "carrier effect" by conjugating purified capsular polymers to carrier proteins to create more effective immunogenic compositions (Schneerson et al. (1984) Infect. Immun. 45:582-591). Conjugation has also been shown to circumvent the poor antibody responses typically observed in infants when immunized with free polysaccharide (Anderson et al. (1985) J. Pediatr. 107:346; Insel et al. (1986) J. Exp. Med. 158:294).

[0004] Conjugates have been successfully produced using a variety of cross-linking or coupling reagents, including homobifunctional, heterobifunctional, or zero-length cross-linking agents. Many methods are currently available for coupling immunogenic molecules, such as sugars, proteins, and peptides, to peptide or protein carriers. Most methods result in amine, amide, urethane, isothiourea, or disulfide bonds, or in some cases, thioethers. A disadvantage of using cross-linking or coupling reagents that introduce reactive sites into the side chains of reactive amino acid molecules on the carrier and / or immunogenic molecule is that, unless neutralized, the reactive sites are free to react with any undesired molecules, either in vitro (thus potentially adversely affecting the functionality or stability of the conjugate) or in vivo (thus posing a potential risk of adverse events in humans or animals immunized with the preparation). Such excess reactive sites can be reacted or "capped" using various known chemical reactions to inactivate these sites, but these reactions can otherwise be destructive to the functionality of the conjugate. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there remains a need for novel glycoconjugates that are appropriately capped so that functionality is preserved and the conjugates retain their ability to elicit a desired immune response, and methods for preparing said conjugates. [Means for solving the problem]

[0006] The present inventors have discovered a novel, effective method for producing glycoconjugates. The method allows for the production of conjugates with very low free sugars and good yields. Furthermore, the present inventors have found that some Streptococcus pneumoniae serotypes (e.g., 35B and 29 polysaccharides) pose particular challenges for producing conjugates due to their unique structure. There is a need for immunogenic Streptococcus pneumoniae serotype polysaccharide-carrier protein conjugates and improved processes for producing the same.

[0007] Summary of the Invention In one aspect, the present invention provides a method for making a capsular glycoglycoconjugate, comprising the steps of: (a) reacting the isolated capsular saccharide with a carbonic acid derivative and an azido linker in an aprotic solvent to produce an activated azido saccharide; (b) reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, wherein the NHS moiety reacts with the amino group to form an amide bond, thereby yielding an alkyne-functionalized carrier protein; (c) Cu +1 reacting the activated azido sugar of step (a) with the activated alkyne carrier protein of step (b) via a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate; The present invention relates to a method comprising:

[0008] In one aspect, the isolated saccharide is sized prior to the activation step (a).

[0009] In one embodiment, the carbonic acid derivative is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.

[0010] In one embodiment, the azide linker has the formula (I):

[0011] [ka] [Wherein X is CH2(CH2) n , (CH2CH2O) m CH2CH2, NHCO(CH2) n , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n , and O(CH2CH2O) m CH2CH2, n is selected from 1 to 10, and m is selected from 1 to 4.

[0012] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

[0013] In one aspect, step a) comprises reacting the capsular saccharide with a carbonate derivative, followed by reacting the carbonate-activated capsular saccharide with an azido linker in an aprotic solvent to produce an activated azido capsular saccharide.

[0014] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst.

[0015] In one aspect, after step c), the method further comprises the step of capping any remaining unreacted azide groups in the conjugate with an azide group capping agent.

[0016] In one aspect, after step c), the method further comprises the step of capping any remaining unreacted alkyne groups in the conjugate with an alkyne group capping agent.

[0017] In one aspect, the invention relates to capsular glycoconjugates produced according to the above method.

[0018] In a further aspect, the present invention comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer and having the general formula (VII):

[0019] [ka] [Wherein X is CH2(CH2) n’ , (CH2CH2O) m CH2CH2, NHCO(CH2) n’ , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ , and O(CH2CH2O) m CH2CH2, n' is selected from 1 to 10, and m is selected from 1 to 4; X' is CH2O(CH2) n” CH2C=O, CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, n" is selected from the group consisting of 0-10, and m' is selected from 0-4.

[0020] In a particular aspect, the present invention comprises a capsular saccharide covalently conjugated to a carrier protein (CP) through a spacer, the capsular saccharide having the general formula (VII), wherein X is CH2(CH2) n’ where n' is 2 and X' is CH2O(CH2) n” CH2C=O and n" is 1.

[0021] In a particular aspect, the present invention comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer, and having the general formula (VIII):

[0022] [ka] It is associated with capsular glycoconjugates having [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the repetitive polysaccharide structure of S. pneumoniae serotype 35B capsular polysaccharide. [Figure 2] FIG. 1 shows the repetitive polysaccharide structure of S. pneumoniae serotype 29 capsular polysaccharide. [Figure 3] FIG. 1 shows a general scheme for the preparation of glycoconjugates of the invention prepared using click chemistry and 3-azido-1-propylamine as the azido linker. CP = carrier protein, CDI = 1,1′-carbonyldiimidazole. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention The present inventors have developed novel polysaccharide-carrier protein conjugates and processes for making these conjugates. They have found that the method for making glycoconjugates is capable of producing glycoconjugates with very low free sugars and good yields.

[0025] Furthermore, the present inventors have found that Streptococcus pneumoniae serotype 35B and 29 polysaccharides pose particular challenges for producing conjugates. They have been found to be cleaved during activation with periodate, a classical oxidizing agent used in commonly used reductive amination processes. Periodate oxidation appears to occur in the backbone of serotype 35B and 29 polysaccharides, cleaving mannitol or ribitol and resulting in size reduction. This leads to several problems, including difficulty in obtaining activated serotype 35B and 29 polysaccharides of a certain size. Periodate activation results in a decrease in the Mw of the polysaccharide, significantly limiting the effective activation range.

[0026] WO2020 / 247299 suggests reducing the amount of periodate and using a specific temperature range for the conjugation reaction to allegedly improve the properties of serotype 35B conjugates. However, such solutions have been found to result in low yields and / or high levels of free sugars. The present inventors have developed novel Streptococcus pneumoniae serotype 35B and 29 polysaccharide-carrier protein conjugates and improved processes for making these conjugates that do not suffer from these drawbacks.

[0027] 1. The glycoconjugate of the present invention The present invention is directed in part to conjugated bacterial capsular saccharide antigens (also called glycoconjugates). For the purposes of the present invention, the term "glycoconjugate" refers to a capsular saccharide (particularly a bacterial capsular saccharide) that is covalently linked to a carrier protein.

[0028] 1.1 Capsular saccharides of the invention The term "saccharide" throughout this specification can refer to either polysaccharides or oligosaccharides, and includes both. In one embodiment, the saccharide of the present invention can be an oligosaccharide. Oligosaccharides have a low number of repeating units (typically 5-15 repeating units) and are typically derived synthetically or by hydrolysis of polysaccharides. Preferably, however, all saccharides in the saccharides of the present invention and immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides are capable of inducing certain antibody immune responses due to epitopes present on the antigen surface. Isolation and purification of high molecular weight capsular polysaccharides are preferably contemplated for use in the conjugates, compositions, and methods of the present invention. Thus, in a preferred embodiment of the present invention, the saccharide is a polysaccharide.

[0029] Preferably, the saccharide used in the present invention is a bacterial capsular saccharide (also referred to herein as "capsular saccharide"). Capsules are found in several medically important bacteria. Bacterial capsules are composed mostly of polysaccharides. Capsular saccharides are prepared by standard techniques known to those skilled in the art.

[0030] In a most preferred embodiment of the invention, the saccharide is a S. pneumoniae capsular polysaccharide.

[0031] In one embodiment, the capsular saccharide for use in the present invention is a synthetic carbohydrate.

[0032] However, in a preferred embodiment, the source of bacterial capsular saccharides according to the present invention may be bacterial cells. Bacterial strains that can be used as a source of capsular saccharides may be obtained from established culture collections (such as the American Type Culture Collection (ATCC), Manassas, Virginia, USA) or the Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, Georgia, USA)) or from clinical specimens.

[0033] Bacterial capsular saccharides can be obtained directly from bacteria using isolation procedures known to those skilled in the art (see, for example, the methods disclosed in US2006 / 0228380, US2006 / 0228381, US2007 / 0184071, US2007 / 0184072, US2007 / 0231340, and US2008 / 0102498, and WO2008 / 118752). They can also be produced using synthetic protocols known to those skilled in the art.

[0034] When bacterial capsular saccharides are obtained directly from bacteria, bacterial cells can be grown in culture media. Following fermentation of the bacterial cells to produce the capsular saccharide, the bacterial cells can be lysed to produce a cell lysate. The capsular saccharide can then be isolated from the cell lysate using purification techniques known in the art (see, for example, US2006 / 0228380, US2006 / 0228381, and WO2008 / 118752), including the use of centrifugation, depth filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography. The purified capsular saccharide can then be used to prepare an immunogenic conjugate.

[0035] The isolated capsular saccharides obtained by purification can be characterized by various parameters, including, for example, weight average molecular weight (Mw), which can be measured by size exclusion chromatography (SEC) coupled with multi-angle laser light scattering detection (MALLS).

[0036] In one embodiment the capsular saccharide used in the method of making a glycoconjugate of the invention or part thereof is a capsular saccharide from a pathogenic bacterium. Preferably the capsular saccharide used in the invention is from a pathogenic Streptococcus, pathogenic Staphylococcus, pathogenic Enterococcus, pathogenic Bacillus, pathogenic Corynebacterium, pathogenic Listeria, pathogenic Erysipelothrix, pathogenic Clostridium, pathogenic Haemophilus, pathogenic Neisseria or pathogenic Escherichia. More preferably, the capsular saccharide for use in the present invention is a capsular saccharide from a pathogenic Streptococcus, a pathogenic Neisseria or a pathogenic Escherichia.

[0037] In one embodiment, the capsular saccharide for use in the invention is selected from the group consisting of Aeromonas hydrophila and other spp.; Bacillus anthracis; Bacillus cereus; Botulinum neurotoxin-producing species of Clostridium; Brucella abortus; Brucella melitensis; Brucella suis; Burkholderia mallei (formerly Pseudomonas mallei); Burkholderia pseudomallei (formerly Pseudomonas pseudomallei); Campylobacter jejuni; jejuni; Chlamydia psittaci; Chlamydia trachomatis; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (heartwater disease); Coxiella burnetii; Enterococcus faecalis;Enterotoxigenic Escherichia coli (EEC group) such as enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC)-O157:H7, and enteroinvasive Escherichia coli (EIEC); Ehrlichia spp. such as Ehrlichia chajfeensis; Francisella tularensis; Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus; Listeria monocytogenes; monocytogenes; miscellaneous enterobacteria such as Klebsiella, Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, and Serratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasma capricolum; Mycoplasma mycoides ssp mycoides; Peronosclerospora philippinensis; Phakopsora pachyrhizi pachyrhizi; Plesiomonas shigelloides; Ralstonia solanacearum race 3, biovar 2; Rickettsia prowazekii;Rickettsia rickettsii, Salmonella spp., Schlerophthora rayssiae var zeae, Shigella spp., Staphylococcus aureus, Streptococcus spp., Synchytrium endobioticum, Vibrio cholerae non-01, Vibrio cholerae 01, Vibrio parahaemolyticus and other Vibrios, Vibrio vulnificus, Xanthomonas oryzae oryzae; Xylella fastidiosa (a strain of citrus variegated yellows); Yersinia enterocolitica and Yersinia pseudotuberculosis; or Yersinia pestis. Preferably, the capsular saccharide used in the present invention is a capsular saccharide from Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, or Streptococcus.

[0038] In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Haemophilus influenzae, Neisseria meningitidis, S. pneumoniae, S. pyogenes, S. agalactiae, Group C and G Streptococci or Escherichia coli. More preferably, the capsular saccharide for use in the invention is a capsular saccharide from Neisseria meningitidis, S. pneumoniae, S. agalactiae or Escherichia coli. Even more preferably, the capsular saccharide for use in the invention is a capsular saccharide from S. pneumoniae or S. agalactiae. Even more preferably, the capsular saccharide for use in the present invention is a capsular saccharide from S. pneumoniae.

[0039] In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Staphylococcus aureus, hi one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Staphylococcus aureus type 5 or Staphylococcus aureus type 8.

[0040] In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Enterococcus faecalis, hi a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Haemophilus influenzae type b.

[0041] In a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Neisseria meningitidis. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup A (MenA), N. meningitidis serogroup W135 (MenW135), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup X (MenX) or N. meningitidis serogroup C (MenC). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup A (MenA). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup W135 (MenW135). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup Y (MenY). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup C (MenC). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from N. meningitidis serogroup X (MenX).

[0042] In a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli.In a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Enterococcus faecalis.

[0043] In a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus agalactiae (Group B Streptococcus (GBS)). In some embodiments, the capsular saccharide for use in the invention is a capsular saccharide from GBS types Ia, Ib, II, III, IV, V, VI, VII or VIII. In some embodiments, the capsular saccharide for use in the invention is a capsular saccharide from GBS types Ia, Ib, II, III or V.

[0044] In a further embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from a part of Escherichia coli of the enterotoxigenic Escherichia coli group (EEC group), such as enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli-O157:H7 (EHEC), or enteroinvasive Escherichia coli (EIEC). In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from uropathogenic Escherichia coli (UPEC).

[0045] In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting of serotypes O157:H7, O26:H11, O111:H- and O103:H2. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1:H7. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting of serotypes O45:K1, O17:K52:H18, O19:H34 and O7:K1. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli serotype O104:H4. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli serotype O1:K12:H7. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli serotype O127:H6. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli serotype O139:H28. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Escherichia coli serotype O128:H2.

[0046] In a preferred embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae. Preferably, the capsular saccharide for use in the invention is from a Streptococcus pneumoniae serotype selected from the group consisting of serotypes 1, 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72 and 73.

[0047] In a preferred embodiment, the capsular saccharide for use in the present invention is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0048] In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 1. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 2. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 4. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 5. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 6A. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 6B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 7C. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 7F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 8. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 9V. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 9N. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 10A. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 10B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 11A.In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 12F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 14. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 15A. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 15B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 15C. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 16F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 17F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 18C. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 19A. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 19F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 20. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 21. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 22A.In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 22F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 23A. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 23B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 23F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 24B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 24F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 27. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 29. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 31. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 33B. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 33F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 34. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 35B.In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 35F. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 38. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 72. In one embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 73.

[0049] In a preferred embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 35B. In another preferred embodiment, the capsular saccharide for use in the invention is a capsular saccharide from Streptococcus pneumoniae serotype 29.

[0050] In a preferred embodiment, the capsular saccharides for use in the invention (purified before further processing) have a weight average molecular weight of between 50 kDa and 5000 kDa. In a preferred embodiment, the capsular saccharides for use in the invention have a weight average molecular weight of between 500 kDa and 5000 kDa. In another preferred embodiment, the capsular saccharides for use in the invention have a weight average molecular weight of between 1000 kDa and 5000 kDa.

[0051] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0052] Preferably, sizing of the saccharide to a target molecular weight range is performed prior to conjugation to the carrier protein to generate glycoconjugates with advantageous filterability characteristics, immunogenicity, and / or yield.

[0053] Advantageously, the purified capsular saccharide is reduced in size while preserving critical features of the polysaccharide structure. Mechanical or chemical sizing may be used.

[0054] In one embodiment, the size of the purified capsular saccharide is reduced by chemical hydrolysis. Chemical hydrolysis may be carried out using a mild acid (e.g., acetic acid, formic acid, propanoic acid). In one embodiment, chemical hydrolysis is carried out using formic acid. In one embodiment, chemical hydrolysis is carried out using propanoic acid. In a preferred embodiment, chemical hydrolysis is carried out using acetic acid. Chemical hydrolysis may also be carried out using a dilute strong acid (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid). In one embodiment, chemical hydrolysis is carried out using dilute hydrochloric acid. In one embodiment, chemical hydrolysis is carried out using dilute sulfuric acid. In one embodiment, chemical hydrolysis is carried out using dilute phosphoric acid. In one embodiment, chemical hydrolysis is carried out using dilute nitric acid. In one embodiment, chemical hydrolysis is carried out using dilute perchloric acid.

[0055] The size of the purified capsular saccharide can also be reduced by mechanical homogenization. In one embodiment, the size of the purified capsular saccharide is reduced by high-pressure homogenization, which achieves a high shear rate by pumping the process stream through a channel with sufficiently small dimensions. The shear rate can be increased by using a greater homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0056] A high pressure homogenisation process may be suitable to reduce the size of purified capsular saccharides whilst preserving the structural characteristics of the saccharide.

[0057] In a preferred embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 10 kDa and 1000 kDa. In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 50 kDa and 500 kDa. In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 50 kDa and 400 kDa. In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 50 kDa and 250 kDa.

[0058] In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 250 kDa and 1000 kDa. In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 250 kDa and 500 kDa. In one embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 250 kDa and 400 kDa. In a preferred embodiment, the isolated capsular saccharides are sized to a weight average molecular weight of between 200 kDa and 800 kDa.

[0059] In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 250 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 300 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 350 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 400 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 450 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 500 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 550 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 600 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 700 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 800 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 900 kDa. In one embodiment, the isolated capsular saccharide is sized to a weight average molecular weight of about 1000 kDa.

[0060] In one embodiment, the isolated capsular saccharide is not sized.

[0061] As described further herein, the isolated capsular saccharides described above may be activated (e.g. chemically activated) to enable them to react (e.g. with a linker) and subsequently be incorporated into glycoconjugates.

[0062] For the purposes of the present invention, the term "glycoconjugate" refers to a sugar that is covalently linked to a carrier protein.

[0063] In general, covalent conjugation of saccharides to carriers enhances the immunogenicity of saccharides by converting them from T-independent antigens to T-dependent antigens, thus enabling priming for immunological memory. Conjugation is particularly useful for pediatric vaccines.

[0064] 1.2 Capsular glycoconjugates of the present invention In some embodiments, the glycoconjugates of the invention comprise capsular saccharides, wherein the weight average molecular weight (Mw) of said polysaccharides prior to conjugation is between 10 kDa and 2,000 kDa.

[0065] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the saccharide (i.e., after the final sizing step, but before the saccharide is reacted with an activating agent). In the context of the present invention, the Mw of the saccharide is not substantially altered by the activation step, and the Mw of the saccharide incorporated into the conjugate is similar to the Mw of the saccharide measured prior to activation. In one embodiment, the saccharide is activated using a carbonic acid derivative (e.g., CDI or CDT) in combination with an azide linker (see Section 1.3, below). In one embodiment, the saccharide is activated using CDI in combination with an azide linker (see Section 1.3, below). In one embodiment, the saccharide is activated using CDT in combination with an azide linker (see Section 1.3, below).

[0066] In one embodiment, the glycoconjugate of the invention comprises a capsular saccharide, wherein said saccharide prior to conjugation has a weight average molecular weight (Mw) of 50 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 250 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 150 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 100 kDa.

[0067] In one embodiment, the glycoconjugates of the invention comprise capsular saccharides, wherein said saccharides prior to conjugation have a weight average molecular weight (Mw) of 75 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 250 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 150 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 100 kDa.

[0068] In one embodiment, the glycoconjugates of the invention comprise capsular saccharides, wherein said saccharides prior to conjugation have a weight average molecular weight (Mw) of 100 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 250 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 150 kDa.

[0069] In one embodiment, the glycoconjugates of the invention comprise capsular saccharides, wherein said saccharides prior to conjugation have a weight average molecular weight (Mw) of between 150 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 250 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 200 kDa.

[0070] In one embodiment, the glycoconjugates of the invention comprise capsular saccharides, wherein said saccharides prior to conjugation have a weight average molecular weight (Mw) of between 200 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 250 kDa.

[0071] In one embodiment, the glycoconjugates of the invention comprise capsular saccharides, wherein said saccharides prior to conjugation have a weight average molecular weight (Mw) of between 500 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 500 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 500 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 500 kDa and 600 kDa.

[0072] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0073] In one embodiment, the glycoconjugates of the invention comprise a capsular saccharide, wherein said saccharide has a weight average molecular weight (Mw) of about 1,000 kDa prior to conjugation. In one embodiment, the weight average molecular weight (Mw) is about 750 kDa. In one embodiment, the weight average molecular weight (Mw) is about 700 kDa. In one embodiment, the weight average molecular weight (Mw) is about 600 kDa. In one embodiment, the weight average molecular weight (Mw) is about 500 kDa. In one embodiment, the weight average molecular weight (Mw) is about 400 kDa. In one embodiment, the weight average molecular weight (Mw) is about 300 kDa. In one embodiment, the weight average molecular weight (Mw) is about 200 kDa. In one embodiment, the weight average molecular weight (Mw) is about 150 kDa. In one embodiment, the weight average molecular weight (Mw) is about 125 kDa. In one embodiment, the weight average molecular weight (Mw) is about 100 kDa.

[0074] In some embodiments, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 250 kDa to 20,000 kDa. In other embodiments, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 15,000 kDa. In yet other embodiments, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 10,000 kDa.

[0075] In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 10,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 7,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 5,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 2,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 2,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 1,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 1,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 500 kDa to 750 kDa.

[0076] In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 10,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 7,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 5,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 2,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 2,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 1,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 750 kDa to 1,000 kDa.

[0077] In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 10,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 7,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 5,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 2,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 2,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 1,500 kDa.

[0078] In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 10,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 7,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 5,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 4,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 3,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,000 kDa to 3,500 kDa.

[0079] In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 2,250 kDa to 3,500 kDa.

[0080] In a preferred embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of 1,000 kDa to 2,500 kDa.

[0081] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0082] In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 10,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 9,000 kDa. In another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 8,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 7,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 6,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 5,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 4,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 3,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 3,250 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 3,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 2,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 2,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 1,500 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 1,000 kDa. In yet another embodiment, the glycoconjugates of the present invention have a weight average molecular weight (Mw) of about 750 kDa.

[0083] Another method for characterizing the glycoconjugates of the present invention is to characterize them by binding to a carrier protein (e.g., a CRMP). 197The degree of conjugation of a glycoconjugate of the present invention depends on the number of lysine residues in the carrier protein (or SCP) that are conjugated to a sugar, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of a carrier protein due to covalent attachment of a sugar can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduced number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated material. In a preferred embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 15. In one embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 13. In one embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 10. In one embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 8. In one embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 6. In one embodiment, the degree of conjugation of a glycoconjugate of the present invention is 2 to 5. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 2 to 4. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 15. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 13. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 10. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 8. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 6. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 5. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 3 to 4. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 5 to 15. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 5 to 10. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 8 to 15. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 8 to 12. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 10 to 15. In one embodiment, the conjugation degree of the glycoconjugate of the present invention is 10 to 12.

[0084] In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 2. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 3. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 4. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 5. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 6. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 7. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 8. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 9. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 10, about 11. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 12. In one embodiment, the conjugation degree of the glycoconjugates of the present invention is about 13. In one embodiment, the degree of conjugation of the glycoconjugates of the present invention is about 14. In one embodiment, the degree of conjugation of the glycoconjugates of the present invention is about 15. In a preferred embodiment, the degree of conjugation of the glycoconjugates of the present invention is between 4 and 7. In some such embodiments, the carrier protein is a CRM. 197 In other such embodiments, the carrier protein is SCP.

[0085] The glycoconjugates of the present invention may also be characterized by the sugar-to-carrier protein ratio (w / w). In some embodiments, the sugar-to-carrier protein ratio (w / w) in the glycoconjugate is 0.5 to 3.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5 to 2.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5 to 1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.8 to 1.2. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5 to 1.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0 to 1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0 to 2.0. In a further embodiment, the sugar-to-carrier protein ratio (w / w) is 0.8 to 1.2. In a preferred embodiment, the ratio of sugar to carrier protein in the conjugate is between 0.9 and 1.1.

[0086] In one embodiment, the ratio of sugar to carrier protein (w / w) is about 0.5. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 0.6. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 0.7. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 0.8. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 0.9. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.0. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.1. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.2. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.3. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.4. In another embodiment, the ratio of sugar to carrier protein (w / w) is about 1.5. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 1.6. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 1.7. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 1.8. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 1.9. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 2.0. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 2.1. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 2.2. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 2.5. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 2.8. In other embodiments, the ratio of sugar to carrier protein (w / w) is about 3.0. In some such embodiments, the carrier protein is CRM. 197 In other such embodiments, the carrier protein is SCP.

[0087] The glycoconjugates of the invention can also be characterized by the number of covalent bonds between the carrier protein and the saccharide as a function of the repeating units of the saccharide. In one embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every four saccharide repeating units of the saccharide. In another embodiment, a covalent bond between the carrier protein and the saccharide occurs at least once for every ten saccharide repeating units of the saccharide. In another embodiment, a covalent bond between the carrier protein and the saccharide occurs at least once for every fifteen saccharide repeating units of the saccharide. In a further embodiment, a covalent bond between the carrier protein and the saccharide occurs at least once for every twenty-five saccharide repeating units of the saccharide. In a further embodiment, a covalent bond between the carrier protein and the saccharide occurs at least once for every fifteen ... hundred saccharide repeating units of the saccharide.

[0088] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 5-10 saccharide repeat units of the saccharide.

[0089] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 2-7 saccharide repeat units of the saccharide.

[0090] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 6-11 saccharide repeat units of the saccharide.

[0091] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 9-14 saccharide repeat units of the saccharide.

[0092] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 10-20 saccharide repeat units of the saccharide.

[0093] In another embodiment, the glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the saccharide for every 4 to 25 saccharide repeat units of the saccharide.

[0094] In frequent embodiments, the carrier protein is a CRM 197 In another embodiment, the carrier protein is SCP.

[0095] In some embodiments, the carrier protein is a CRM 197 and CRM 197 and the covalent bond between the SCP and the saccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeat units of the saccharide. In other embodiments, the carrier protein is an SCP and the covalent bond between the SCP and the saccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeat units of the saccharide.

[0096] The glycoconjugates and immunogenic compositions of the invention may contain free saccharides that are not covalently conjugated to a carrier protein but are still present in the glycoconjugate composition. The free saccharide may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound to, adsorbed to, or encapsulated in or with it).

[0097] In a preferred embodiment, the complex carbohydrate comprises less than about 50% free sugars relative to the total amount of said sugars. In a preferred embodiment, the complex carbohydrate comprises less than about 40% free sugars relative to the total amount of said sugars. In a further preferred embodiment, the complex carbohydrate comprises less than about 25% free sugars relative to the total amount of said sugars. In a further preferred embodiment, the complex carbohydrate comprises less than about 20% free sugars relative to the total amount of said sugars. In a further preferred embodiment, the complex carbohydrate comprises less than about 15% free sugars relative to the total amount of said sugars.

[0098] Complex carbohydrates also vary in their molecular size distribution (K d) can also be characterized. Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in gravity-fed columns to profile the molecular size distribution of the conjugates. Large molecules in the media that are excluded from the pores elute earlier than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically by sugar assay. K d To determine the fraction, the column is calibrated to determine the fraction at which the molecule is completely excluded (V0), (K d = 0), and the fraction representing maximum retention (V i ), (K d Establish the fraction (V = 1) at which a specified sample characteristic is reached. e ) is the formula, K d =(V e -V0) / (V i -V0), K d Related to.

[0099] In a preferred embodiment, at least 30% of the glycoconjugates of the invention have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 40% of the glycoconjugates of the present invention have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the glycoconjugates of the invention have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 60% of the glycoconjugates of the present invention have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 50% to 80% of the glycoconjugates of the present invention have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 65% to 80% of the glycoconjugates of the present invention have a K of 0.3 or less in a CL-4B column. d It has.

[0100] 1.3 Capsular glycoconjugates of the invention prepared using click chemistry The glycoconjugates of the present invention are prepared using click chemistry. The present invention also relates to methods of making the glycoconjugates as disclosed herein.

[0101] According to the present invention, click chemistry involves three steps: (a) reacting isolated capsular saccharides with a carbonic acid derivative and an azido linker in an aprotic solvent to produce an activated azido saccharide (saccharide activation); (b) reacting a carrier protein with a drug bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group, where the NHS moiety reacts with the amino group to form an amide bond, thereby yielding an alkyne-functionalized carrier protein (carrier protein activation); and (c) reacting Cu +1 and reacting the activated azido sugar of step (a) with the activated alkyne carrier protein of step (b) via a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate.

[0102] After step (a), the sugar is said to be activated and is referred to herein as an "activated sugar" or an "activated azido sugar."

[0103] After step (b), the support is said to be activated and is called an "activated support."

[0104] As described above, activation (a) can be preceded by sizing the sugar to a target molecular weight (MW) range. Thus, in one embodiment, the isolated sugar is sized prior to activation with a carbonate derivative and an azide linker. In one embodiment, the isolated sugar is sized to any of the target molecular weight (MW) ranges defined above.

[0105] In one embodiment, the carbonic acid derivative is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.

[0106] In one embodiment, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI). In another embodiment, the carbonic acid derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In another embodiment, the carbonic acid derivative is disuccinimidyl carbonate (DSC). In a further embodiment, the carbonic acid derivative is N-hydroxysuccinimidyl chloroformate.

[0107] In one embodiment, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI) or 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). Preferably, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI).

[0108] In one embodiment, the azide linker has formula (I):

[0109] [ka] [Wherein X is CH2(CH2) n , (CH2CH2O) m CH2CH2, NHCO(CH2) n , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n , and O(CH2CH2O) m CH2CH2, n is selected from 1 to 10, and m is selected from 1 to 4.

[0110] In one embodiment, the azido linker has formula (I), wherein X is CH2(CH2) nand n is selected from 1 to 10. In one embodiment, n is selected from 1 to 5. In one embodiment, n is selected from 1 to 4. In one embodiment, n is selected from 1 to 3. In one embodiment, n is selected from 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10.

[0111] In one embodiment, the azido linker has formula (I), wherein X is (CH2CH2O) m CH2CH2, and m is selected from 1 to 4. In one embodiment, m is selected from 1 to 3. In one embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In a further embodiment, m is 4.

[0112] In one embodiment, the azido linker has formula (I), wherein X is NHCO(CH) n and n is selected from 1 to 10. In one embodiment, n is selected from 1 to 5. In one embodiment, n is selected from 1 to 4. In one embodiment, n is selected from 1 to 3. In one embodiment, n is selected from 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10.

[0113] In one embodiment, the azido linker has formula (I), wherein X is NHCO(CH2CH2O)m CH2CH2, and m is selected from 1 to 4. In one embodiment, m is selected from 1 to 3. In one embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In a further embodiment, m is 4.

[0114] In one embodiment, the azido linker has formula (I), wherein X is OCH2(CH2) n and n is selected from 1 to 10. In one embodiment, n is selected from 1 to 5. In one embodiment, n is selected from 1 to 4. In one embodiment, n is selected from 1 to 3. In one embodiment, n is selected from 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10.

[0115] In one embodiment, the azido linker has formula (I), wherein X is O(CH2CH2O) m CH2CH2, and m is selected from 1 to 4. In one embodiment, m is selected from 1 to 3. In one embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In a further embodiment, m is 4.

[0116] In one embodiment, the azido linker has formula (II):

[0117] [ka] is a compound of

[0118] In a preferred embodiment, the azido linker is 3-azido-propylamine.

[0119] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

[0120] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne.

[0121] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has formula (III):

[0122] [ka] [Wherein X is CH2O(CH2) n CH2C=O and CHO(CH2CH2O) m (CH2) n CH2C=O, n is selected from 0 to 10, and m is selected from 0 to 4.

[0123] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has the formula (III), wherein X is CHO(CH) nCH2C=O, and n is selected from 0-10. In one embodiment, n is selected from 0-5. In one embodiment, n is selected from 0-4. In one embodiment, n is selected from 0-3. In one embodiment, n is selected from 0-2. In a particular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10.

[0124] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has the formula (III), wherein X is CHO(CHCHO) m (CH2) n CH2C=O, n is selected from 0-10, and m is selected from 0-4. In one embodiment, n is selected from 0-5. In one embodiment, n is selected from 0-4. In one embodiment, n is selected from 0-3. In one embodiment, n is selected from 0-2. In certain embodiments, n is 0. In certain embodiments, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10. In one embodiment, m is selected from 0-3. In one embodiment, m is selected from 0-2. In certain embodiments, m is 1. In certain embodiments, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In a further embodiment, m is 4.

[0125] In one embodiment, n is selected from 0 to 5, and m is selected from 0 to 3. In one embodiment, n is selected from 0 to 5, and m is selected from 0 to 2.

[0126] In one embodiment, n is selected from 0 to 4, and m is selected from 0 to 3. In one embodiment, n is selected from 0 to 4, and m is selected from 0 to 2.

[0127] In one embodiment, n is selected from 0 to 3, and m is selected from 0 to 3. In one embodiment, n is selected from 0 to 3, and m is selected from 0 to 2.

[0128] In one embodiment, n is selected from 0 to 2 and m is selected from 0 to 3. In one embodiment, n is selected from 0 to 2 and m is selected from 0 to 2.

[0129] In one embodiment, n is selected from 0 to 1 and m is selected from 0 to 3. In one embodiment, n is selected from 0 to 1 and m is selected from 0 to 2.

[0130] In one embodiment, n is 0 and m is 0. In one embodiment, n is 1 and m is 0. In one embodiment, n is 2 and m is 0. In one embodiment, n is 3 and m is 0. In one embodiment, n is 4 and m is 0. In one embodiment, n is 5 and m is 0. In one embodiment, n is 6 and m is 0. In one embodiment, n is 7 and m is 0. In one embodiment, n is 8 and m is 0. In one embodiment, n is 9 and m is 0. In one embodiment, n is 10 and m is 0.

[0131] In one embodiment, n is 0 and m is 1. In one embodiment, n is 1 and m is 1. In one embodiment, n is 2 and m is 1. In one embodiment, n is 3 and m is 1. In one embodiment, n is 4 and m is 1. In one embodiment, n is 5 and m is 1. In one embodiment, n is 6 and m is 1. In one embodiment, n is 7 and m is 1. In one embodiment, n is 8 and m is 1. In one embodiment, n is 9 and m is 1. In one embodiment, n is 10 and m is 1.

[0132] In one embodiment, n is 0 and m is 2. In one embodiment, n is 1 and m is 2. In one embodiment, n is 2 and m is 2. In one embodiment, n is 3 and m is 2. In one embodiment, n is 4 and m is 2. In one embodiment, n is 5 and m is 2. In one embodiment, n is 6 and m is 2. In one embodiment, n is 7 and m is 2. In one embodiment, n is 8 and m is 2. In one embodiment, n is 9 and m is 2. In one embodiment, n is 10 and m is 2.

[0133] In one embodiment, n is 0 and m is 3. In one embodiment, n is 1 and m is 3. In one embodiment, n is 2 and m is 3. In one embodiment, n is 3 and m is 3. In one embodiment, n is 4 and m is 3. In one embodiment, n is 5 and m is 3. In one embodiment, n is 6 and m is 3. In one embodiment, n is 7 and m is 3. In one embodiment, n is 8 and m is 3. In one embodiment, n is 9 and m is 3. In one embodiment, n is 10 and m is 3.

[0134] In one embodiment, n is 0 and m is 4. In one embodiment, n is 1 and m is 4. In one embodiment, n is 2 and m is 4. In one embodiment, n is 3 and m is 4. In one embodiment, n is 4 and m is 4. In one embodiment, n is 5 and m is 4. In one embodiment, n is 6 and m is 4. In one embodiment, n is 7 and m is 4. In one embodiment, n is 8 and m is 4. In one embodiment, n is 9 and m is 4. In one embodiment, n is 10 and m is 4.

[0135] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has formula (IV):

[0136] [ka] is a compound of

[0137] In one embodiment, step a) comprises reacting a sugar with a carbonic acid derivative, followed by reacting the carbonic acid-activated sugar with an azido linker in an aprotic solvent to produce an activated azido sugar.

[0138] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0139] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0140] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0141] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0142] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is between 0.3 and 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0143] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0144] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.5 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0145] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.8 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0146] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 1 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0147] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is between 2 and 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0148] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is between 3 and 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0149] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 5 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0150] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0151] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0152] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0153] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0154] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0155] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0156] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.5 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0157] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.8 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0158] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0159] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 2 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0160] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 3 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0161] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0162] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0163] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0164] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0165] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0166] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0167] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.5 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0168] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.8 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0169] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 1 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0170] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 2 to 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0171] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0172] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0173] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0174] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0175] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0176] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0177] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.5 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0178] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.8 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0179] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 1 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0180] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0181] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0182] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0183] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0184] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0185] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0186] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.5 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0187] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.8 to 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0188] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0189] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0190] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0191] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0192] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0193] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.4 to 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0194] In one embodiment, step a) comprises reacting a sugar with an amount of a carbonic acid derivative.

[0195] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0196] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0197] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0198] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0199] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.3 to 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0200] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.01 to 0.3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0201] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.05 to 0.3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0202] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.1 to 0.3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0203] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is 0.2 to 0.3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0204] In one embodiment, step a) comprises reacting the sugar with an amount of the carbonic acid derivative that is about 0.01 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0205] In one embodiment, step a) comprises reacting the sugar with an amount of the carbonic acid derivative that is about 0.05 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0206] In one embodiment, step a) comprises reacting the sugar with an amount of about 0.08 molar equivalents of the carbonic acid derivative relative to the amount of sugar present in the reaction mixture.

[0207] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 0.1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0208] In one embodiment, step a) comprises reacting the sugar with an amount of about 0.2 molar equivalents of the carbonic acid derivative relative to the amount of sugar present in the reaction mixture.

[0209] In one embodiment, step a) comprises reacting the sugar with an amount of about 0.3 molar equivalents of the carbonic acid derivative relative to the amount of sugar present in the reaction mixture.

[0210] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 0.4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0211] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0212] In one embodiment, step a) comprises reacting the sugar with about 1 molar equivalent of the carbonic acid derivative relative to the amount of sugar present in the reaction mixture.

[0213] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0214] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 3 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0215] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 4 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0216] In one embodiment, step a) comprises reacting the sugar with an amount of the carbonic acid derivative that is about 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0217] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 8 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0218] In one embodiment, step a) comprises reacting the sugar with an amount of carbonic acid derivative that is about 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0219] In one embodiment, in step a), the isolated sugar is reacted with a carbonic acid derivative in an aprotic solvent.

[0220] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution consisting essentially of dimethyl formamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0221] In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in a solution consisting essentially of dimethylacetamide. In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in a solution consisting essentially of N-methyl-2-pyrrolidone. In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in a solution consisting essentially of hexamethylphosphoramide (HMPA).

[0222] In a preferred embodiment, the isolated sugar is reacted with a carbonic acid derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0223] In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl formamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO).

[0224] In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in dimethylacetamide. In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in N-methyl-2-pyrrolidone. In one embodiment, the isolated sugar is reacted with a carbonic acid derivative in hexamethylphosphoramide (HMPA).

[0225] In a preferred embodiment, the isolated sugar is reacted with CDI in dimethyl sulfoxide (DMSO). In one embodiment, the isolated sugar is reacted with CDI in anhydrous DMSO.

[0226] Surprisingly, it has been found that side reactions can be avoided by reacting the isolated sugar with CDI in an environment having a moisture level of about 0.1% to 1% (v / v). Thus, in one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.3% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.2% (v / v) water.

[0227] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.3% (v / v) water.

[0228] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.4% (v / v) water.

[0229] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.2% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.3% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.6% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent comprising about 0.7% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.9% (v / v) water.

[0230] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.3% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.2% (v / v) water.

[0231] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.3% (v / v) water.

[0232] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.4% (v / v) water.

[0233] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.2% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.3% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.6% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.7% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.8% (v / v) water. In one embodiment, the isolated saccharide is reacted with CDI in DMSO containing about 0.9% (v / v) water.

[0234] In one embodiment, the free carbonic acid derivative is then quenched by adding water before adding the azide linker. Water can inactivate the free CDI. Thus, in one embodiment, activation of the carbonic acid derivative is followed by the addition of water. In one embodiment, water is added to bring the total water content in the mixture to about 1% to about 10% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.2% to about 8% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.5% to about 5% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.5% to about 3% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.5% to about 2.5% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.2% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.4% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 1.5% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 2% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 2.5% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 3% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 5% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 7% (v / v). In one embodiment, water is added to bring the total water content in the mixture to about 10% (v / v).

[0235] After reacting the sugar with a carbonate derivative and final quenching of the carbonate derivative with water, the carbonate-activated sugar is reacted with an azide linker.

[0236] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 10 molar equivalents relative to the amount of polysaccharide repeating units (molar equivalents of RU) of the activated saccharide.

[0237] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 8 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0238] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0239] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0240] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0241] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0242] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0243] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0244] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.01 to 0.1 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0245] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0246] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0247] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0248] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0249] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 3 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0250] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0251] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0252] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 0.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0253] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.05 to 0.1 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0254] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0255] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0256] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0257] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0258] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 3 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0259] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0260] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0261] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.1 to 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0262] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0263] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0264] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0265] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0266] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0267] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0268] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0269] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0270] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0271] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0272] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0273] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0274] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 1 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0275] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 2 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0276] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 2 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0277] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 2 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0278] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 2 to 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0279] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 2 to 3 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0280] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 3 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0281] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 3 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0282] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 3 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0283] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 3 to 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0284] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 4 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0285] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 4 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0286] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 4 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0287] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 5 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0288] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 5 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0289] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is 8 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0290] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 0.01 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0291] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 0.05 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0292] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 0.1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0293] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0294] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated saccharide.

[0295] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0296] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0297] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0298] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0299] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0300] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated saccharide with an amount of azide linker that is about 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0301] In the above embodiment, the carbonate derivative is preferably CDI. In another embodiment, the carbonate derivative is CDT.

[0302] In one embodiment, the degree of activation of the activated sugar after step a) is 0.5-50%. The degree of activation of the azido sugar is defined as the percentage of repeating units linked with an azido linker.

[0303] In one embodiment, the degree of activation of the activated sugar after step a) is 1-30%. In another embodiment, the degree of activation of the activated sugar after step a) is 2-25%. In another embodiment, the degree of activation of the activated sugar after step a) is 3-20%.

[0304] In another embodiment, the degree of activation of the activated sugar after step a) is 3-15%. In another embodiment, the degree of activation of the activated sugar after step a) is 4-15%. In one embodiment, the degree of activation of the activated sugar after step a) is 1-6%.

[0305] In one embodiment, the degree of activation of the activated sugar after step a) is 3-6%. In one embodiment, the degree of activation of the activated sugar after step a) is 10-15%.

[0306] In one embodiment, the degree of activation of the activated sugar after step a) is about 1%. In one embodiment, the degree of activation of the activated sugar after step a) is about 2%. In one embodiment, the degree of activation of the activated sugar after step a) is about 3%. In one embodiment, the degree of activation of the activated sugar after step a) is about 4%. In one embodiment, the degree of activation of the activated sugar after step a) is about 5%. In one embodiment, the degree of activation of the activated sugar after step a) is about 6%. In one embodiment, the degree of activation of the activated sugar after step a) is about 7%. In one embodiment, the degree of activation of the activated sugar after step a) is about 8%. In one embodiment, the degree of activation of the activated sugar after step a) is about 9%. In one embodiment, the degree of activation of the activated sugar after step a) is about 10%. In one embodiment, the degree of activation of the activated sugar after step a) is about 11%. In one embodiment, the degree of activation of the activated sugar after step a) is about 12%. In one embodiment, the degree of activation of the activated sugar after step a) is about 13%. In one embodiment, the degree of activation of the activated sugar after step a) is about 14%. In one embodiment, the degree of activation of the activated sugar after step a) is about 15%. In one embodiment, the degree of activation of the activated sugar after step a) is about 16%. In one embodiment, the degree of activation of the activated sugar after step a) is about 17%. In one embodiment, the degree of activation of the activated sugar after step a) is about 18%. In one embodiment, the degree of activation of the activated sugar after step a) is about 19%. In one embodiment, the degree of activation of the activated sugar after step a) is about 20%.

[0307] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 10 molar equivalents relative to the lysines on the carrier.

[0308] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 10 molar equivalents relative to the lysines on the carrier.

[0309] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 10 molar equivalents relative to the lysines on the carrier.

[0310] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 10 molar equivalents relative to the lysines on the carrier.

[0311] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2 to 10 molar equivalents relative to the lysines on the carrier.

[0312] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5 to 10 molar equivalents relative to the lysines on the carrier.

[0313] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3 to 10 molar equivalents relative to the lysines on the carrier.

[0314] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 5 to 10 molar equivalents relative to the lysines on the carrier.

[0315] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 7.5 to 10 molar equivalents relative to the lysines on the carrier.

[0316] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 7.5 molar equivalents relative to the lysines on the carrier.

[0317] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 7.5 molar equivalents relative to the lysines on the carrier.

[0318] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 7.5 molar equivalents relative to the lysines on the carrier.

[0319] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 7.5 molar equivalents relative to the lysines on the carrier.

[0320] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2 to 7.5 molar equivalents relative to the lysines on the carrier.

[0321] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5 to 7.5 molar equivalents relative to the lysines on the carrier.

[0322] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3 to 7.5 molar equivalents relative to the lysines on the carrier.

[0323] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 5 to 7.5 molar equivalents relative to the lysines on the carrier.

[0324] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 5 molar equivalents relative to the lysines on the carrier.

[0325] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 5 molar equivalents relative to the lysines on the carrier.

[0326] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 5 molar equivalents relative to the lysines on the carrier.

[0327] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 5 molar equivalents relative to the lysines on the carrier.

[0328] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2 to 5 molar equivalents relative to the lysines on the carrier.

[0329] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5 to 5 molar equivalents relative to the lysines on the carrier.

[0330] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3 to 5 molar equivalents relative to the lysines on the carrier.

[0331] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 3 molar equivalents relative to the lysines on the carrier.

[0332] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 3 molar equivalents relative to the lysines on the carrier.

[0333] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 3 molar equivalents relative to the lysines on the carrier.

[0334] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 3 molar equivalents relative to the lysines on the carrier.

[0335] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2-3 molar equivalents relative to the lysines on the carrier.

[0336] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5 to 3 molar equivalents relative to the lysines on the carrier.

[0337] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 2.5 molar equivalents relative to the lysines on the carrier.

[0338] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 2.5 molar equivalents relative to the lysines on the carrier.

[0339] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 2.5 molar equivalents relative to the lysines on the carrier.

[0340] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 2.5 molar equivalents relative to the lysines on the carrier.

[0341] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2 to 2.5 molar equivalents relative to the lysines on the carrier.

[0342] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 2 molar equivalents relative to the lysines on the carrier.

[0343] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 2 molar equivalents relative to the lysines on the carrier.

[0344] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 2 molar equivalents relative to the lysines on the carrier.

[0345] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5 to 2 molar equivalents relative to the lysines on the carrier.

[0346] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 1.5 molar equivalents relative to the lysines on the carrier.

[0347] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 1.5 molar equivalents relative to the lysines on the carrier.

[0348] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1 to 1.5 molar equivalents relative to the lysines on the carrier.

[0349] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 1 molar equivalent relative to the lysines on the carrier.

[0350] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5 to 1 molar equivalent relative to the lysines on the carrier.

[0351] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1 to 0.5 molar equivalents relative to the lysines on the carrier.

[0352] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 10 molar equivalents relative to the lysines on the carrier.

[0353] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 7.5 molar equivalents relative to the lysines on the carrier.

[0354] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 5 molar equivalents relative to the lysines on the carrier.

[0355] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 3 molar equivalents relative to the lysines on the carrier.

[0356] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 2.5 molar equivalents relative to the lysines on the carrier.

[0357] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 2 molar equivalents relative to the lysines on the carrier.

[0358] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 1.5 molar equivalents relative to the lysines on the carrier.

[0359] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 1 molar equivalent relative to the lysines on the carrier.

[0360] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 0.5 molar equivalents relative to the lysines on the carrier.

[0361] In one embodiment, step b) comprises reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 0.1 molar equivalent relative to the lysines on the carrier.

[0362] In one embodiment, the activation degree of the activated carrier after step b) is between 1 and 50. The activation degree of the activated carrier is defined as the number of lysine residues in the carrier protein that are linked to a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group.

[0363] In one embodiment, the carrier protein is a CRM containing 39 lysine residues. 197 In the above embodiment, the activation degree of the activating support after step b) can be 1 to 30. In another embodiment, the activation degree of the activating support (CRM) after step b) can be 1 to 30. 197In another embodiment, the activation degree of the activating carrier (CRM) after step b) is 5 to 20. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is 9 to 18. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is 8 to 11. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is 15 to 20. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 5. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 6. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 7. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 8. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 9. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 10. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 11. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 12. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 13. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 14. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 15. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 16. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 17. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 18.197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 19. 197 In another embodiment, the activation degree of the activating carrier (CRM) after step b) is about 20. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 21. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 22. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 23. 197 In another embodiment, the activation degree of the activating support (CRM) after step b) is about 24. 197 ) has an activation level of about 25.

[0364] In one embodiment, the carrier protein is an SCP or a fragment thereof. In said embodiment, the activation degree of the activating carrier after step b) may be 1 to 50. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 5 to 50. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 7 to 45. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 5 to 15. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 20 to 30. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 30 to 50. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 30 to 40. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is 10 to 40. In another embodiment, the activation degree of the activating carrier (SCP) after step b) is about 5. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 7. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 10. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 13. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 15. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 20. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 26. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 30. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 35. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 37. In another embodiment, the activation degree of the activating support (SCP) after step b) is about 40. In another embodiment, the degree of activation of the activating support (SCP) after step b) is about 45. In another embodiment, the degree of activation of the activating support (SCP) after step b) is about 50.

[0365] In one embodiment, the carrier protein is TT or a fragment thereof. In said embodiment, the activation degree of the activating carrier after step b) may be 1 to 30. In another embodiment, the activation degree of the activating carrier (TT) after step b) is 5 to 25. In another embodiment, the activation degree of the activating carrier (TT) after step b) is 7 to 25. In another embodiment, the activation degree of the activating carrier (TT) after step b) is 10 to 20. In another embodiment, the activation degree of the activating carrier (TT) after step b) is about 5. In another embodiment, the activation degree of the activating carrier (TT) after step b) is about 7. In another embodiment, the activation degree of the activating carrier (TT) after step b) is about 10. In another embodiment, the activation degree of the activating carrier (TT) after step b) is about 12. In another embodiment, the activation degree of the activating carrier (TT) after step b) is about 15. In another embodiment, the degree of activation of the activating support (TT) after step b) is about 20. In another embodiment, the degree of activation of the activating support (TT) after step b) is about 25. In another embodiment, the degree of activation of the activating support (TT) after step b) is about 30.

[0366] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst. In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of an oxidizing agent and copper(I) as a catalyst. In a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst and ascorbate as an oxidizing agent. In one embodiment, THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine may be further added to protect the protein from side reactions. Thus, in a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer solution in the presence of copper(I) as a catalyst and ascorbate as an oxidizing agent, and the reaction mixture further comprises THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine.

[0367] In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is 0.1 to 3. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is 0.5 to 2. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is 0.6 to 1.5. In a preferred embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is 0.8 to 1. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 0.5. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 0.6. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 0.7. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 0.8. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 0.9. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.1. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.2. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.3. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.4. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.5. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.6.In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.7. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.8. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 1.9. In one embodiment, the initial input ratio (weight to weight) of activated azido sugar to activated alkyne support in step c) is about 2.

[0368] After the click conjugation reaction, unreacted azide groups may remain in the conjugate, which can be capped using an appropriate azide group capping agent. Thus, in one embodiment, after step c), the unreacted azide groups in the conjugate are capped using an appropriate azide group capping agent. In one embodiment, the azide group capping agent is an agent having an alkyne group. In one embodiment, the azide group capping agent is an agent having a terminal alkyne. In one embodiment, the azide group capping agent is an agent having a cycloalkyne.

[0369] In one embodiment, the azide group capping agent has formula (V):

[0370] [ka] [Wherein X is (CH2) n and n is selected from 1 to 15.

[0371] In one embodiment, the azide group capping agent is propargyl alcohol.

[0372] Thus, in one embodiment, after step (c), the process further comprises capping any remaining unreacted azide groups in the conjugate with an azide group capping agent.

[0373] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.05 to 20 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0374] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.1 to 15 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0375] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0376] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0377] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0378] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated saccharide.

[0379] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 1-2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0380] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 0.75 to 1.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0381] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is about 1 molar equivalent to the amount of polysaccharide repeating units of the activated saccharide.

[0382] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is about 1.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0383] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is about 0.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0384] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is about 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0385] After the click conjugation reaction, there may be unreacted alkyne groups remaining in the conjugate, which can be capped using a suitable alkyne group capping agent, which in one embodiment is an agent bearing an azide group.

[0386] In one embodiment, the alkyne group capping agent has formula (VI):

[0387] [ka] [Wherein X is (CH2) n and n is selected from 1 to 15.

[0388] In one embodiment, the alkyne group capping agent is 3-azido-1-propanol.

[0389] Thus, in one embodiment, after step (c), the process further comprises the step of capping any remaining unreacted alkyne groups in the conjugate with an alkyne group capping agent.

[0390] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.05 to 20 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0391] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.1 to 15 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0392] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0393] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0394] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0395] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated saccharide.

[0396] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0397] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1-2 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0398] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1.5 to 2.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated saccharide.

[0399] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0400] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 1 molar equivalent to the amount of polysaccharide repeat units of the activated saccharide.

[0401] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 1.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0402] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0403] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 2.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0404] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is about 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated saccharide.

[0405] After conjugation with the carrier protein, the glycoconjugates can be purified (enriched in terms of the amount of glyco-protein conjugates) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. Thus, in one embodiment, the process for producing the glycoconjugates of the present invention includes a step of purifying the glycoconjugates after they have been produced.

[0406] In one aspect, the invention provides glycoconjugates produced according to any of the methods disclosed herein.

[0407] In one aspect, the present invention comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer and having the general formula (VII):

[0408] [ka] [Wherein X is CH2(CH2) n’ , (CH2CH2O) m CH2CH2, NHCO(CH2) n’ , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ , and O(CH2CH2O) m CH2CH2, n' is selected from 1 to 10, and m is selected from 1 to 4; X' is CH2O(CH2) n” CH2C=O, CH2O(CH2CH2O) m’ (CH2) n” CHC=O, n" is selected from the group consisting of 0-10, and m' is selected from 0-4, wherein the structure in square brackets represents the repeating unit of the capsular saccharide.

[0409] In one embodiment the capsular saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0410] Formula (VII) is a schematic representation of a glycoconjugate of the invention. It should not be understood that linkages exist at all repeating units of the saccharide (structures in square brackets). Rather, the majority of the saccharide repeating units are left unmodified, and covalent bonds between the carrier protein and the saccharide are present at a minority of the saccharide repeating units. Furthermore, a single carrier protein (CP) molecule may be linked to multiple saccharide molecules, and a single saccharide molecule can be linked to more than one carrier protein (CP) molecule. The structure in square brackets represents the repeating unit of a capsular saccharide.

[0411] In a preferred embodiment, the present invention comprises a saccharide covalently conjugated to a carrier protein (CP) through a spacer, the saccharide having the general formula (VII), wherein X is CH2(CH2) n’ where n' is 2 and X' is CH2O(CH2) n”CH2C═O and n″ is 1. In one embodiment, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0412] Thus, in a preferred embodiment, the present invention comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer, and having the general formula (VIII):

[0413] [ka] The present invention provides a capsular glycoconjugate having the following structure:

[0414] Here, the structure in square brackets represents the repeating unit of the capsular saccharide.

[0415] Formula (VIII) is a schematic representation of a preferred glycoconjugate of the present invention. It should not be understood that linkages exist at all repeating units of the sugar (structure in square brackets). Rather, the majority of the sugar repeating units are left unmodified, and the covalent bond between the carrier protein and the sugar is at a minority of the sugar repeating units. Furthermore, a single carrier protein (CP) molecule may be linked to multiple sugar molecules, and a single sugar molecule may be linked to more than one carrier protein (CP) molecule. The structure in square brackets is a schematic representation of the repeating units of the sugar.

[0416] In one embodiment the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0417] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is CH2(CH2) n’ and n' is selected from 1 to 10, and X' is CHO(CH) n”CH2C═O and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-5. In one embodiment, n′ is selected from 1-3 and n″ is selected from 0-3. In one embodiment, n′ is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, n′ is 1 and n″ is 0. In another embodiment, n′ is 2 and n″ is 0. In yet another embodiment, n′ is 3 and n″ is 0. In a further embodiment, n′ is 4 and n″ is 0. In a further embodiment, n′ is 5 and n″ is 0. In a further embodiment, n′ is 6 and n″ is 0. In a particular embodiment, n′ is 1 and n″ is 1. In another embodiment, n' is 2 and n" is 1. In yet another embodiment, n' is 3 and n" is 1. In a further embodiment, n' is 4 and n" is 1. In a further embodiment, n' is 5 and n" is 1. In a further embodiment, n' is 6 and n" is 1. In a particular embodiment, n' is 1 and n" is 2. In another embodiment, n' is 2 and n" is 2. In yet another embodiment, n' is 3 and n" is 2. In a further embodiment, n' is 4 and n" is 2. In a further embodiment, n' is 5 and n" is 2. In a further embodiment, n' is 6 and n" is 2. In a particular embodiment, n' is 1 and n" is 3. In another embodiment, n' is 2 and n" is 3. In yet another embodiment, n' is 3 and n" is 3. In a further embodiment, n' is 4 and n" is 3. In a further embodiment, n' is 5 and n" is 3. In a further embodiment, n' is 6 and n" is 3. In a particular embodiment, n' is 1 and n" is 4. In another embodiment, n' is 2 and n" is 4. In yet another embodiment, n' is 3 and n" is 4. In a further embodiment, n' is 4 and n" is 4. In a further embodiment, n' is 5 and n" is 4. In a further embodiment, n' is 6 and n" is 4.In a particular embodiment, n' is 1 and n" is 5. In another embodiment, n' is 2 and n" is 5. In yet another embodiment, n' is 3 and n" is 5. In a further embodiment, n' is 4 and n" is 5. In a further embodiment, n' is 5 and n" is 5. In a further embodiment, n' is 6 and n" is 5. In a particular embodiment, n' is 1 and n" is 6. In another embodiment, n' is 2 and n" is 6. In yet another embodiment, n' is 3 and n" is 6. In a further embodiment, n' is 4 and n" is 6. In a further embodiment, n' is 5 and n" is 6. In a further embodiment, n' is 6 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0418] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is CH2(CH2) n’ and n' is selected from 1 to 10, and X' is CHO(CHCHO) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0419] In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0420] In certain embodiments, n' is 1, m' is 0, and n" is 0. In other embodiments, n' is 1, m' is 1, and n" is 0. In other embodiments, n' is 1, m' is 2, and n" is 0. In other embodiments, n' is 1, m' is 3, and n" is 0.

[0421] In another embodiment, n' is 2, m' is 0, and n" is 0. In another embodiment, n' is 2, m' is 1, and n" is 0. In another embodiment, n' is 2, m' is 2, and n" is 0. In another embodiment, n' is 2, m' is 3, and n" is 0.

[0422] In yet another embodiment, n' is 3, m' is 0, and n" is 0. In yet another embodiment, n' is 3, m' is 1, and n" is 0. In yet another embodiment, n' is 3, m' is 2, and n" is 0. In yet another embodiment, n' is 3, m' is 3, and n" is 0.

[0423] In a further embodiment, n' is 4, m' is 0, and n" is 0. In a further embodiment, n' is 4, m' is 1, and n" is 0. In a further embodiment, n' is 4, m' is 2, and n" is 0. In a further embodiment, n' is 4, m' is 3, and n" is 0.

[0424] In a further embodiment, n' is 5, m' is 0, and n" is 0. In a further embodiment, n' is 5, m' is 1, and n" is 0. In a further embodiment, n' is 5, m' is 2, and n" is 0. In a further embodiment, n' is 5, m' is 3, and n" is 0.

[0425] In certain embodiments, n' is 1, m' is 0, and n" is 1. In certain embodiments, n' is 1, m' is 1, and n" is 1. In certain embodiments, n' is 1, m' is 2, and n" is 1. In certain embodiments, n' is 1, m' is 3, and n" is 1.

[0426] In another embodiment, n' is 2, m' is 0, and n" is 1. In another embodiment, n' is 2, m' is 1, and n" is 1. In another embodiment, n' is 2, m' is 2, and n" is 1. In another embodiment, n' is 2, m' is 3, and n" is 1.

[0427] In yet another embodiment, n' is 3, m' is 0, and n" is 1. In yet another embodiment, n' is 3, m' is 1, and n" is 1. In yet another embodiment, n' is 3, m' is 2, and n" is 1. In yet another embodiment, n' is 3, m' is 3, and n" is 1.

[0428] In a further embodiment, n' is 4, m' is 0, and n" is 1. In a further embodiment, n' is 4, m' is 1, and n" is 1. In a further embodiment, n' is 4, m' is 2, and n" is 1. In a further embodiment, n' is 4, m' is 3, and n" is 1.

[0429] In a further embodiment, n' is 5, m' is 0, and n" is 1. In a further embodiment, n' is 5, m' is 1, and n" is 1. In a further embodiment, n' is 5, m' is 2, and n" is 1. In a further embodiment, n' is 5, m' is 3, and n" is 1.

[0430] In certain embodiments, n' is 1, m' is 0, and n" is 2. In certain embodiments, n' is 1, m' is 1, and n" is 2. In certain embodiments, n' is 1, m' is 2, and n" is 2. In certain embodiments, n' is 1, m' is 3, and n" is 2.

[0431] In another embodiment, n' is 2, m' is 0, and n" is 2. In another embodiment, n' is 2, m' is 1, and n" is 2. In another embodiment, n' is 2, m' is 2, and n" is 2. In another embodiment, n' is 2, m' is 3, and n" is 2.

[0432] In yet another embodiment, n' is 3, m' is 0, and n" is 2. In yet another embodiment, n' is 3, m' is 1, and n" is 2. In yet another embodiment, n' is 3, m' is 2, and n" is 2. In yet another embodiment, n' is 3, m' is 3, and n" is 2.

[0433] In a further embodiment, n' is 4, m' is 0, and n" is 2. In a further embodiment, n' is 4, m' is 1, and n" is 2. In a further embodiment, n' is 4, m' is 2, and n" is 2. In a further embodiment, n' is 4, m' is 3, and n" is 2.

[0434] In a further embodiment, n' is 5, m' is 0, and n" is 2. In a further embodiment, n' is 5, m' is 1, and n" is 2. In a further embodiment, n' is 5, m' is 2, and n" is 2. In a further embodiment, n' is 5, m' is 3, and n" is 2.

[0435] In certain embodiments, n' is 1, m' is 0, and n" is 3. In certain embodiments, n' is 1, m' is 1, and n" is 3. In certain embodiments, n' is 1, m' is 2, and n" is 3. In certain embodiments, n' is 1, m' is 3, and n" is 3.

[0436] In another embodiment, n' is 2, m' is 0, and n" is 3. In another embodiment, n' is 2, m' is 1, and n" is 3. In another embodiment, n' is 2, m' is 2, and n" is 3. In another embodiment, n' is 2, m' is 3, and n" is 3.

[0437] In yet another embodiment, n' is 3, m' is 0, and n" is 3. In yet another embodiment, n' is 3, m' is 1, and n" is 3. In yet another embodiment, n' is 3, m' is 2, and n" is 3. In yet another embodiment, n' is 3, m' is 3, and n" is 3.

[0438] In a further embodiment, n' is 4, m' is 0, and n" is 3. In a further embodiment, n' is 4, m' is 1, and n" is 3. In a further embodiment, n' is 4, m' is 2, and n" is 3. In a further embodiment, n' is 4, m' is 3, and n" is 3.

[0439] In a further embodiment, n' is 5, m' is 0, and n" is 3. In a further embodiment, n' is 5, m' is 1, and n" is 3. In a further embodiment, n' is 5, m' is 2, and n" is 3. In a further embodiment, n' is 5, m' is 3, and n" is 3.

[0440] In certain embodiments, n' is 1, m' is 0, and n" is 4. In certain embodiments, n' is 1, m' is 1, and n" is 4. In certain embodiments, n' is 1, m' is 2, and n" is 4. In certain embodiments, n' is 1, m' is 3, and n" is 4.

[0441] In another embodiment, n' is 2, m' is 0, and n" is 4. In another embodiment, n' is 2, m' is 1, and n" is 4. In another embodiment, n' is 2, m' is 2, and n" is 4. In another embodiment, n' is 2, m' is 3, and n" is 4.

[0442] In yet another embodiment, n' is 3, m' is 0, and n" is 4. In yet another embodiment, n' is 3, m' is 1, and n" is 4. In yet another embodiment, n' is 3, m' is 2, and n" is 4. In yet another embodiment, n' is 3, m' is 3, and n" is 4.

[0443] In a further embodiment, n' is 4, m' is 0, and n" is 4. In a further embodiment, n' is 4, m' is 1, and n" is 4. In a further embodiment, n' is 4, m' is 2, and n" is 4. In a further embodiment, n' is 4, m' is 3, and n" is 4.

[0444] In a further embodiment, n' is 5, m' is 0, and n" is 4. In a further embodiment, n' is 5, m' is 1, and n" is 4. In a further embodiment, n' is 5, m' is 2, and n" is 4. In a further embodiment, n' is 5, m' is 3, and n" is 4.

[0445] In certain embodiments, n' is 1, m' is 0, and n" is 5. In certain embodiments, n' is 1, m' is 1, and n" is 5. In certain embodiments, n' is 1, m' is 2, and n" is 5. In certain embodiments, n' is 1, m' is 3, and n" is 5.

[0446] In another embodiment, n' is 2, m' is 0, and n" is 5. In another embodiment, n' is 2, m' is 1, and n" is 5. In another embodiment, n' is 2, m' is 2, and n" is 5. In another embodiment, n' is 2, m' is 3, and n" is 5.

[0447] In yet another embodiment, n' is 3, m' is 0, and n" is 5. In yet another embodiment, n' is 3, m' is 1, and n" is 5. In yet another embodiment, n' is 3, m' is 2, and n" is 5. In yet another embodiment, n' is 3, m' is 3, and n" is 5.

[0448] In a further embodiment, n' is 4, m' is 0, and n" is 5. In a further embodiment, n' is 4, m' is 1, and n" is 5. In a further embodiment, n' is 4, m' is 2, and n" is 5. In a further embodiment, n' is 4, m' is 3, and n" is 5.

[0449] In a further embodiment, n' is 5, m' is 0, and n" is 5. In a further embodiment, n' is 5, m' is 1, and n" is 5. In a further embodiment, n' is 5, m' is 2, and n" is 5. In a further embodiment, n' is 5, m' is 3, and n" is 5.

[0450] Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0451] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is (CH2CH2O) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n”CH2C═O and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-5. In one embodiment, m is selected from 1-2 and n″ is selected from 0-3. In one embodiment, m is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, m is 1 and n″ is 0. In another embodiment, m is 2 and n″ is 0. In yet another embodiment, m is 3 and n″ is 0. In a further embodiment, m is 4 and n″ is 0. In a particular embodiment, m is 1 and n″ is 1. In another embodiment, m is 2 and n″ is 1. In yet another embodiment, m is 3 and n″ is 1. In further embodiments, m is 4 and n" is 1. In certain embodiments, m is 1 and n" is 2. In another embodiment, m is 2 and n" is 2. In yet another embodiment, m is 3 and n" is 2. In further embodiments, m is 4 and n" is 2. In certain embodiments, m is 1 and n" is 3. In another embodiment, m is 2 and n" is 3. In yet another embodiment, m is 3 and n" is 3. In further embodiments, m is 4 and n" is 3. In certain embodiments, m is 1 and n" is 4. In another embodiment, m is 2 and n" is 4. In yet another embodiment, m is 3 and n" is 4. In further embodiments, m is 4 and n" is 4. In certain embodiments, m is 1 and n" is 5. In another embodiment, m is 2 and n" is 5. In yet another embodiment, m is 3 and n" is 5. In a further embodiment, m is 4 and n" is 5. In a particular embodiment, m is 1 and n" is 6. In another embodiment, m is 2 and n" is 6. In yet another embodiment, m is 3 and n" is 6. In a further embodiment, m is 4 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0452] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is (CH2CH2O) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0453] In one embodiment, m is selected from 1 to 3, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0454] In certain embodiments, m is 1, m' is 0, and n" is 0. In other embodiments, m is 1, m' is 1, and n" is 0. In other embodiments, m is 1, m' is 2, and n" is 0. In other embodiments, m is 1, m' is 3, and n" is 0.

[0455] In another embodiment, m is 2, m' is 0, and n" is 0. In another embodiment, m is 2, m' is 1, and n" is 0. In another embodiment, m is 2, m' is 2, and n" is 0. In another embodiment, m is 2, m' is 3, and n" is 0.

[0456] In yet another embodiment, m is 3, m' is 0, and n" is 0. In yet another embodiment, m is 3, m' is 1, and n" is 0. In yet another embodiment, m is 3, m' is 2, and n" is 0. In yet another embodiment, m is 3, m' is 3, and n" is 0.

[0457] In a further embodiment, m is 4, m' is 0, and n" is 0. In a further embodiment, m is 4, m' is 1, and n" is 0. In a further embodiment, m is 4, m' is 2, and n" is 0. In a further embodiment, m is 4, m' is 3, and n" is 0.

[0458] In certain embodiments, m is 1, m' is 0, and n" is 1. In certain embodiments, m is 1, m' is 1, and n" is 1. In certain embodiments, m is 1, m' is 2, and n" is 1. In certain embodiments, m is 1, m' is 3, and n" is 1.

[0459] In another embodiment, m is 2, m' is 0, and n" is 1. In another embodiment, m is 2, m' is 1, and n" is 1. In another embodiment, m is 2, m' is 2, and n" is 1. In another embodiment, m is 2, m' is 3, and n" is 1.

[0460] In yet another embodiment, m is 3, m' is 0, and n" is 1. In yet another embodiment, m is 3, m' is 1, and n" is 1. In yet another embodiment, m is 3, m' is 2, and n" is 1. In yet another embodiment, m is 3, m' is 3, and n" is 1.

[0461] In a further embodiment, m is 4, m' is 0, and n" is 1. In a further embodiment, m is 4, m' is 1, and n" is 1. In a further embodiment, m is 4, m' is 2, and n" is 1. In a further embodiment, m is 4, m' is 3, and n" is 1.

[0462] In certain embodiments, m is 1, m' is 0, and n" is 2. In certain embodiments, m is 1, m' is 1, and n" is 2. In certain embodiments, m is 1, m' is 2, and n" is 2. In certain embodiments, m is 1, m' is 3, and n" is 2.

[0463] In another embodiment, m is 2, m' is 0, and n" is 2. In another embodiment, m is 2, m' is 1, and n" is 2. In another embodiment, m is 2, m' is 2, and n" is 2. In another embodiment, m is 2, m' is 3, and n" is 2.

[0464] In yet another embodiment, m is 3, m' is 0, and n" is 2. In yet another embodiment, m is 3, m' is 1, and n" is 2. In yet another embodiment, m is 3, m' is 2, and n" is 2. In yet another embodiment, m is 3, m' is 3, and n" is 2.

[0465] In a further embodiment, m is 4, m' is 0, and n" is 2. In a further embodiment, m is 4, m' is 1, and n" is 2. In a further embodiment, m is 4, m' is 2, and n" is 2. In a further embodiment, m is 4, m' is 3, and n" is 2.

[0466] In certain embodiments, m is 1, m' is 0, and n" is 3. In certain embodiments, m is 1, m' is 1, and n" is 3. In certain embodiments, m is 1, m' is 2, and n" is 3. In certain embodiments, m is 1, m' is 3, and n" is 3.

[0467] In another embodiment, m is 2, m' is 0, and n" is 3. In another embodiment, m is 2, m' is 1, and n" is 3. In another embodiment, m is 2, m' is 2, and n" is 3. In another embodiment, m is 2, m' is 3, and n" is 3.

[0468] In yet another embodiment, m is 3, m' is 0, and n" is 3. In yet another embodiment, m is 3, m' is 1, and n" is 3. In yet another embodiment, m is 3, m' is 2, and n" is 3. In yet another embodiment, m is 3, m' is 3, and n" is 3.

[0469] In a further embodiment, m is 4, m' is 0, and n" is 3. In a further embodiment, m is 4, m' is 1, and n" is 3. In a further embodiment, m is 4, m' is 2, and n" is 3. In a further embodiment, m is 4, m' is 3, and n" is 3.

[0470] In certain embodiments, m is 1, m' is 0, and n" is 4. In certain embodiments, m is 1, m' is 1, and n" is 4. In certain embodiments, m is 1, m' is 2, and n" is 4. In certain embodiments, m is 1, m' is 3, and n" is 4.

[0471] In another embodiment, m is 2, m' is 0, and n" is 4. In another embodiment, m is 2, m' is 1, and n" is 4. In another embodiment, m is 2, m' is 2, and n" is 4. In another embodiment, m is 2, m' is 3, and n" is 4.

[0472] In yet another embodiment, m is 3, m' is 0, and n" is 4. In yet another embodiment, m is 3, m' is 1, and n" is 4. In yet another embodiment, m is 3, m' is 2, and n" is 4. In yet another embodiment, m is 3, m' is 3, and n" is 4.

[0473] In a further embodiment, m is 4, m' is 0, and n" is 4. In a further embodiment, m is 4, m' is 1, and n" is 4. In a further embodiment, m is 4, m' is 2, and n" is 4. In a further embodiment, m is 4, m' is 3, and n" is 4.

[0474] In certain embodiments, m is 1, m' is 0, and n" is 5. In certain embodiments, m is 1, m' is 1, and n" is 5. In certain embodiments, m is 1, m' is 2, and n" is 5. In certain embodiments, m is 1, m' is 3, and n" is 5.

[0475] In another embodiment, m is 2, m' is 0, and n" is 5. In another embodiment, m is 2, m' is 1, and n" is 5. In another embodiment, m is 2, m' is 2, and n" is 5. In another embodiment, m is 2, m' is 3, and n" is 5.

[0476] In yet another embodiment, m is 3, m' is 0, and n" is 5. In yet another embodiment, m is 3, m' is 1, and n" is 5. In yet another embodiment, m is 3, m' is 2, and n" is 5. In yet another embodiment, m is 3, m' is 3, and n" is 5.

[0477] In a further embodiment, m is 4, m' is 0, and n" is 5. In a further embodiment, m is 4, m' is 1, and n" is 5. In a further embodiment, m is 4, m' is 2, and n" is 5. In a further embodiment, m is 4, m' is 3, and n" is 5.

[0478] Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0479] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is NHCO(CH) n’ and n' is selected from 1 to 10, and X' is CHO(CH) n”CH2C═O and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-5. In one embodiment, n′ is selected from 1-3 and n″ is selected from 0-3. In one embodiment, n′ is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, n′ is 1 and n″ is 0. In another embodiment, n′ is 2 and n″ is 0. In yet another embodiment, n′ is 3 and n″ is 0. In a further embodiment, n′ is 4 and n″ is 0. In a further embodiment, n′ is 5 and n″ is 0. In a further embodiment, n′ is 6 and n″ is 0. In a particular embodiment, n′ is 1 and n″ is 1. In another embodiment, n' is 2 and n" is 1. In yet another embodiment, n' is 3 and n" is 1. In a further embodiment, n' is 4 and n" is 1. In a further embodiment, n' is 5 and n" is 1. In a further embodiment, n' is 6 and n" is 1. In a particular embodiment, n' is 1 and n" is 2. In another embodiment, n' is 2 and n" is 2. In yet another embodiment, n' is 3 and n" is 2. In a further embodiment, n' is 4 and n" is 2. In a further embodiment, n' is 5 and n" is 2. In a further embodiment, n' is 6 and n" is 2. In a particular embodiment, n' is 1 and n" is 3. In another embodiment, n' is 2 and n" is 3. In yet another embodiment, n' is 3 and n" is 3. In a further embodiment, n' is 4 and n" is 3. In a further embodiment, n' is 5 and n" is 3. In a further embodiment, n' is 6 and n" is 3. In a particular embodiment, n' is 1 and n" is 4. In another embodiment, n' is 2 and n" is 4. In yet another embodiment, n' is 3 and n" is 4. In a further embodiment, n' is 4 and n" is 4. In a further embodiment, n' is 5 and n" is 4. In a further embodiment, n' is 6 and n" is 4.In a particular embodiment, n' is 1 and n" is 5. In another embodiment, n' is 2 and n" is 5. In yet another embodiment, n' is 3 and n" is 5. In a further embodiment, n' is 4 and n" is 5. In a further embodiment, n' is 5 and n" is 5. In a further embodiment, n' is 6 and n" is 5. In a particular embodiment, n' is 1 and n" is 6. In another embodiment, n' is 2 and n" is 6. In yet another embodiment, n' is 3 and n" is 6. In a further embodiment, n' is 4 and n" is 6. In a further embodiment, n' is 5 and n" is 6. In a further embodiment, n' is 6 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0480] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is NHCO(CH) n’ and n' is selected from 1 to 10, and X' is CHO(CHCHO) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0481] In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0482] In certain embodiments, n' is 1, m' is 0, and n" is 0. In other embodiments, n' is 1, m' is 1, and n" is 0. In other embodiments, n' is 1, m' is 2, and n" is 0. In other embodiments, n' is 1, m' is 3, and n" is 0.

[0483] In another embodiment, n' is 2, m' is 0, and n" is 0. In another embodiment, n' is 2, m' is 1, and n" is 0. In another embodiment, n' is 2, m' is 2, and n" is 0. In another embodiment, n' is 2, m' is 3, and n" is 0.

[0484] In yet another embodiment, n' is 3, m' is 0, and n" is 0. In yet another embodiment, n' is 3, m' is 1, and n" is 0. In yet another embodiment, n' is 3, m' is 2, and n" is 0. In yet another embodiment, n' is 3, m' is 3, and n" is 0.

[0485] In a further embodiment, n' is 4, m' is 0, and n" is 0. In a further embodiment, n' is 4, m' is 1, and n" is 0. In a further embodiment, n' is 4, m' is 2, and n" is 0. In a further embodiment, n' is 4, m' is 3, and n" is 0.

[0486] In a further embodiment, n' is 5, m' is 0, and n" is 0. In a further embodiment, n' is 5, m' is 1, and n" is 0. In a further embodiment, n' is 5, m' is 2, and n" is 0. In a further embodiment, n' is 5, m' is 3, and n" is 0.

[0487] In certain embodiments, n' is 1, m' is 0, and n" is 1. In certain embodiments, n' is 1, m' is 1, and n" is 1. In certain embodiments, n' is 1, m' is 2, and n" is 1. In certain embodiments, n' is 1, m' is 3, and n" is 1.

[0488] In another embodiment, n' is 2, m' is 0, and n" is 1. In another embodiment, n' is 2, m' is 1, and n" is 1. In another embodiment, n' is 2, m' is 2, and n" is 1. In another embodiment, n' is 2, m' is 3, and n" is 1.

[0489] In yet another embodiment, n' is 3, m' is 0, and n" is 1. In yet another embodiment, n' is 3, m' is 1, and n" is 1. In yet another embodiment, n' is 3, m' is 2, and n" is 1. In yet another embodiment, n' is 3, m' is 3, and n" is 1.

[0490] In a further embodiment, n' is 4, m' is 0, and n" is 1. In a further embodiment, n' is 4, m' is 1, and n" is 1. In a further embodiment, n' is 4, m' is 2, and n" is 1. In a further embodiment, n' is 4, m' is 3, and n" is 1.

[0491] In a further embodiment, n' is 5, m' is 0, and n" is 1. In a further embodiment, n' is 5, m' is 1, and n" is 1. In a further embodiment, n' is 5, m' is 2, and n" is 1. In a further embodiment, n' is 5, m' is 3, and n" is 1.

[0492] In certain embodiments, n' is 1, m' is 0, and n" is 2. In certain embodiments, n' is 1, m' is 1, and n" is 2. In certain embodiments, n' is 1, m' is 2, and n" is 2. In certain embodiments, n' is 1, m' is 3, and n" is 2.

[0493] In another embodiment, n' is 2, m' is 0, and n" is 2. In another embodiment, n' is 2, m' is 1, and n" is 2. In another embodiment, n' is 2, m' is 2, and n" is 2. In another embodiment, n' is 2, m' is 3, and n" is 2.

[0494] In yet another embodiment, n' is 3, m' is 0, and n" is 2. In yet another embodiment, n' is 3, m' is 1, and n" is 2. In yet another embodiment, n' is 3, m' is 2, and n" is 2. In yet another embodiment, n' is 3, m' is 3, and n" is 2.

[0495] In a further embodiment, n' is 4, m' is 0, and n" is 2. In a further embodiment, n' is 4, m' is 1, and n" is 2. In a further embodiment, n' is 4, m' is 2, and n" is 2. In a further embodiment, n' is 4, m' is 3, and n" is 2.

[0496] In a further embodiment, n' is 5, m' is 0, and n" is 2. In a further embodiment, n' is 5, m' is 1, and n" is 2. In a further embodiment, n' is 5, m' is 2, and n" is 2. In a further embodiment, n' is 5, m' is 3, and n" is 2.

[0497] In certain embodiments, n' is 1, m' is 0, and n" is 3. In certain embodiments, n' is 1, m' is 1, and n" is 3. In certain embodiments, n' is 1, m' is 2, and n" is 3. In certain embodiments, n' is 1, m' is 3, and n" is 3.

[0498] In another embodiment, n' is 2, m' is 0, and n" is 3. In another embodiment, n' is 2, m' is 1, and n" is 3. In another embodiment, n' is 2, m' is 2, and n" is 3. In another embodiment, n' is 2, m' is 3, and n" is 3.

[0499] In yet another embodiment, n' is 3, m' is 0, and n" is 3. In yet another embodiment, n' is 3, m' is 1, and n" is 3. In yet another embodiment, n' is 3, m' is 2, and n" is 3. In yet another embodiment, n' is 3, m' is 3, and n" is 3.

[0500] In a further embodiment, n' is 4, m' is 0, and n" is 3. In a further embodiment, n' is 4, m' is 1, and n" is 3. In a further embodiment, n' is 4, m' is 2, and n" is 3. In a further embodiment, n' is 4, m' is 3, and n" is 3.

[0501] In a further embodiment, n' is 5, m' is 0, and n" is 3. In a further embodiment, n' is 5, m' is 1, and n" is 3. In a further embodiment, n' is 5, m' is 2, and n" is 3. In a further embodiment, n' is 5, m' is 3, and n" is 3.

[0502] In certain embodiments, n' is 1, m' is 0, and n" is 4. In certain embodiments, n' is 1, m' is 1, and n" is 4. In certain embodiments, n' is 1, m' is 2, and n" is 4. In certain embodiments, n' is 1, m' is 3, and n" is 4.

[0503] In another embodiment, n' is 2, m' is 0, and n" is 4. In another embodiment, n' is 2, m' is 1, and n" is 4. In another embodiment, n' is 2, m' is 2, and n" is 4. In another embodiment, n' is 2, m' is 3, and n" is 4.

[0504] In yet another embodiment, n' is 3, m' is 0, and n" is 4. In yet another embodiment, n' is 3, m' is 1, and n" is 4. In yet another embodiment, n' is 3, m' is 2, and n" is 4. In yet another embodiment, n' is 3, m' is 3, and n" is 4.

[0505] In a further embodiment, n' is 4, m' is 0, and n" is 4. In a further embodiment, n' is 4, m' is 1, and n" is 4. In a further embodiment, n' is 4, m' is 2, and n" is 4. In a further embodiment, n' is 4, m' is 3, and n" is 4.

[0506] In a further embodiment, n' is 5, m' is 0, and n" is 4. In a further embodiment, n' is 5, m' is 1, and n" is 4. In a further embodiment, n' is 5, m' is 2, and n" is 4. In a further embodiment, n' is 5, m' is 3, and n" is 4.

[0507] In certain embodiments, n' is 1, m' is 0, and n" is 5. In certain embodiments, n' is 1, m' is 1, and n" is 5. In certain embodiments, n' is 1, m' is 2, and n" is 5. In certain embodiments, n' is 1, m' is 3, and n" is 5.

[0508] In another embodiment, n' is 2, m' is 0, and n" is 5. In another embodiment, n' is 2, m' is 1, and n" is 5. In another embodiment, n' is 2, m' is 2, and n" is 5. In another embodiment, n' is 2, m' is 3, and n" is 5.

[0509] In yet another embodiment, n' is 3, m' is 0, and n" is 5. In yet another embodiment, n' is 3, m' is 1, and n" is 5. In yet another embodiment, n' is 3, m' is 2, and n" is 5. In yet another embodiment, n' is 3, m' is 3, and n" is 5.

[0510] In a further embodiment, n' is 4, m' is 0, and n" is 5. In a further embodiment, n' is 4, m' is 1, and n" is 5. In a further embodiment, n' is 4, m' is 2, and n" is 5. In a further embodiment, n' is 4, m' is 3, and n" is 5.

[0511] In a further embodiment, n' is 5, m' is 0, and n" is 5. In a further embodiment, n' is 5, m' is 1, and n" is 5. In a further embodiment, n' is 5, m' is 2, and n" is 5. In a further embodiment, n' is 5, m' is 3, and n" is 5.

[0512] Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0513] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is NHCO(CHCHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n”CH2C═O and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-5. In one embodiment, m is selected from 1-2 and n″ is selected from 0-3. In one embodiment, m is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, m is 1 and n″ is 0. In another embodiment, m is 2 and n″ is 0. In yet another embodiment, m is 3 and n″ is 0. In a further embodiment, m is 4 and n″ is 0. In a particular embodiment, m is 1 and n″ is 1. In another embodiment, m is 2 and n″ is 1. In yet another embodiment, m is 3 and n″ is 1. In further embodiments, m is 4 and n" is 1. In certain embodiments, m is 1 and n" is 2. In another embodiment, m is 2 and n" is 2. In yet another embodiment, m is 3 and n" is 2. In further embodiments, m is 4 and n" is 2. In certain embodiments, m is 1 and n" is 3. In another embodiment, m is 2 and n" is 3. In yet another embodiment, m is 3 and n" is 3. In further embodiments, m is 4 and n" is 3. In certain embodiments, m is 1 and n" is 4. In another embodiment, m is 2 and n" is 4. In yet another embodiment, m is 3 and n" is 4. In further embodiments, m is 4 and n" is 4. In certain embodiments, m is 1 and n" is 5. In another embodiment, m is 2 and n" is 5. In yet another embodiment, m is 3 and n" is 5. In a further embodiment, m is 4 and n" is 5. In a particular embodiment, m is 1 and n" is 6. In another embodiment, m is 2 and n" is 6. In yet another embodiment, m is 3 and n" is 6. In a further embodiment, m is 4 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0514] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is NHCO(CHCHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0515] In one embodiment, m is selected from 1 to 3, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0516] In certain embodiments, m is 1, m' is 0, and n" is 0. In other embodiments, m is 1, m' is 1, and n" is 0. In other embodiments, m is 1, m' is 2, and n" is 0. In other embodiments, m is 1, m' is 3, and n" is 0.

[0517] In another embodiment, m is 2, m' is 0, and n" is 0. In another embodiment, m is 2, m' is 1, and n" is 0. In another embodiment, m is 2, m' is 2, and n" is 0. In another embodiment, m is 2, m' is 3, and n" is 0.

[0518] In yet another embodiment, m is 3, m' is 0, and n" is 0. In yet another embodiment, m is 3, m' is 1, and n" is 0. In yet another embodiment, m is 3, m' is 2, and n" is 0. In yet another embodiment, m is 3, m' is 3, and n" is 0.

[0519] In a further embodiment, m is 4, m' is 0, and n" is 0. In a further embodiment, m is 4, m' is 1, and n" is 0. In a further embodiment, m is 4, m' is 2, and n" is 0. In a further embodiment, m is 4, m' is 3, and n" is 0.

[0520] In certain embodiments, m is 1, m' is 0, and n" is 1. In certain embodiments, m is 1, m' is 1, and n" is 1. In certain embodiments, m is 1, m' is 2, and n" is 1. In certain embodiments, m is 1, m' is 3, and n" is 1.

[0521] In another embodiment, m is 2, m' is 0, and n" is 1. In another embodiment, m is 2, m' is 1, and n" is 1. In another embodiment, m is 2, m' is 2, and n" is 1. In another embodiment, m is 2, m' is 3, and n" is 1.

[0522] In yet another embodiment, m is 3, m' is 0, and n" is 1. In yet another embodiment, m is 3, m' is 1, and n" is 1. In yet another embodiment, m is 3, m' is 2, and n" is 1. In yet another embodiment, m is 3, m' is 3, and n" is 1.

[0523] In a further embodiment, m is 4, m' is 0, and n" is 1. In a further embodiment, m is 4, m' is 1, and n" is 1. In a further embodiment, m is 4, m' is 2, and n" is 1. In a further embodiment, m is 4, m' is 3, and n" is 1.

[0524] In certain embodiments, m is 1, m' is 0, and n" is 2. In certain embodiments, m is 1, m' is 1, and n" is 2. In certain embodiments, m is 1, m' is 2, and n" is 2. In certain embodiments, m is 1, m' is 3, and n" is 2.

[0525] In another embodiment, m is 2, m' is 0, and n" is 2. In another embodiment, m is 2, m' is 1, and n" is 2. In another embodiment, m is 2, m' is 2, and n" is 2. In another embodiment, m is 2, m' is 3, and n" is 2.

[0526] In yet another embodiment, m is 3, m' is 0, and n" is 2. In yet another embodiment, m is 3, m' is 1, and n" is 2. In yet another embodiment, m is 3, m' is 2, and n" is 2. In yet another embodiment, m is 3, m' is 3, and n" is 2.

[0527] In a further embodiment, m is 4, m' is 0, and n" is 2. In a further embodiment, m is 4, m' is 1, and n" is 2. In a further embodiment, m is 4, m' is 2, and n" is 2. In a further embodiment, m is 4, m' is 3, and n" is 2.

[0528] In certain embodiments, m is 1, m' is 0, and n" is 3. In certain embodiments, m is 1, m' is 1, and n" is 3. In certain embodiments, m is 1, m' is 2, and n" is 3. In certain embodiments, m is 1, m' is 3, and n" is 3.

[0529] In another embodiment, m is 2, m' is 0, and n" is 3. In another embodiment, m is 2, m' is 1, and n" is 3. In another embodiment, m is 2, m' is 2, and n" is 3. In another embodiment, m is 2, m' is 3, and n" is 3.

[0530] In yet another embodiment, m is 3, m' is 0, and n" is 3. In yet another embodiment, m is 3, m' is 1, and n" is 3. In yet another embodiment, m is 3, m' is 2, and n" is 3. In yet another embodiment, m is 3, m' is 3, and n" is 3.

[0531] In a further embodiment, m is 4, m' is 0, and n" is 3. In a further embodiment, m is 4, m' is 1, and n" is 3. In a further embodiment, m is 4, m' is 2, and n" is 3. In a further embodiment, m is 4, m' is 3, and n" is 3.

[0532] In certain embodiments, m is 1, m' is 0, and n" is 4. In certain embodiments, m is 1, m' is 1, and n" is 4. In certain embodiments, m is 1, m' is 2, and n" is 4. In certain embodiments, m is 1, m' is 3, and n" is 4.

[0533] In another embodiment, m is 2, m' is 0, and n" is 4. In another embodiment, m is 2, m' is 1, and n" is 4. In another embodiment, m is 2, m' is 2, and n" is 4. In another embodiment, m is 2, m' is 3, and n" is 4.

[0534] In yet another embodiment, m is 3, m' is 0, and n" is 4. In yet another embodiment, m is 3, m' is 1, and n" is 4. In yet another embodiment, m is 3, m' is 2, and n" is 4. In yet another embodiment, m is 3, m' is 3, and n" is 4.

[0535] In a further embodiment, m is 4, m' is 0, and n" is 4. In a further embodiment, m is 4, m' is 1, and n" is 4. In a further embodiment, m is 4, m' is 2, and n" is 4. In a further embodiment, m is 4, m' is 3, and n" is 4.

[0536] In certain embodiments, m is 1, m' is 0, and n" is 5. In certain embodiments, m is 1, m' is 1, and n" is 5. In certain embodiments, m is 1, m' is 2, and n" is 5. In certain embodiments, m is 1, m' is 3, and n" is 5.

[0537] In another embodiment, m is 2, m' is 0, and n" is 5. In another embodiment, m is 2, m' is 1, and n" is 5. In another embodiment, m is 2, m' is 2, and n" is 5. In another embodiment, m is 2, m' is 3, and n" is 5.

[0538] In yet another embodiment, m is 3, m' is 0, and n" is 5. In yet another embodiment, m is 3, m' is 1, and n" is 5. In yet another embodiment, m is 3, m' is 2, and n" is 5. In yet another embodiment, m is 3, m' is 3, and n" is 5.

[0539] In a further embodiment m is 4, m' is 0 and n" is 5. In a further embodiment m is 4, m' is 1 and n" is 5. In a further embodiment m is 4, m' is 2 and n" is 5. In a further embodiment m is 4, m' is 3 and n" is 5. Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0540] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is OCH2(CH2)n’ and n' is selected from 1 to 10, and X' is CHO(CH) n”CH2C═O and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-10. In one embodiment, n′ is selected from 1-5 and n″ is selected from 0-5. In one embodiment, n′ is selected from 1-3 and n″ is selected from 0-3. In one embodiment, n′ is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, n′ is 1 and n″ is 0. In another embodiment, n′ is 2 and n″ is 0. In yet another embodiment, n′ is 3 and n″ is 0. In a further embodiment, n′ is 4 and n″ is 0. In a further embodiment, n′ is 5 and n″ is 0. In a further embodiment, n′ is 6 and n″ is 0. In a particular embodiment, n′ is 1 and n″ is 1. In another embodiment, n' is 2 and n" is 1. In yet another embodiment, n' is 3 and n" is 1. In a further embodiment, n' is 4 and n" is 1. In a further embodiment, n' is 5 and n" is 1. In a further embodiment, n' is 6 and n" is 1. In a particular embodiment, n' is 1 and n" is 2. In another embodiment, n' is 2 and n" is 2. In yet another embodiment, n' is 3 and n" is 2. In a further embodiment, n' is 4 and n" is 2. In a further embodiment, n' is 5 and n" is 2. In a further embodiment, n' is 6 and n" is 2. In a particular embodiment, n' is 1 and n" is 3. In another embodiment, n' is 2 and n" is 3. In yet another embodiment, n' is 3 and n" is 3. In a further embodiment, n' is 4 and n" is 3. In a further embodiment, n' is 5 and n" is 3. In a further embodiment, n' is 6 and n" is 3. In a particular embodiment, n' is 1 and n" is 4. In another embodiment, n' is 2 and n" is 4. In yet another embodiment, n' is 3 and n" is 4. In a further embodiment, n' is 4 and n" is 4. In a further embodiment, n' is 5 and n" is 4. In a further embodiment, n' is 6 and n" is 4.In a particular embodiment, n' is 1 and n" is 5. In another embodiment, n' is 2 and n" is 5. In yet another embodiment, n' is 3 and n" is 5. In a further embodiment, n' is 4 and n" is 5. In a further embodiment, n' is 5 and n" is 5. In a further embodiment, n' is 6 and n" is 5. In a particular embodiment, n' is 1 and n" is 6. In another embodiment, n' is 2 and n" is 6. In yet another embodiment, n' is 3 and n" is 6. In a further embodiment, n' is 4 and n" is 6. In a further embodiment, n' is 5 and n" is 6. In a further embodiment, n' is 6 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0541] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is OCH2(CH2) n’ and n' is selected from 1 to 10, and X' is CHO(CHCHO) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0542] In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0543] In certain embodiments, n' is 1, m' is 0, and n" is 0. In other embodiments, n' is 1, m' is 1, and n" is 0. In other embodiments, n' is 1, m' is 2, and n" is 0. In other embodiments, n' is 1, m' is 3, and n" is 0.

[0544] In another embodiment, n' is 2, m' is 0, and n" is 0. In another embodiment, n' is 2, m' is 1, and n" is 0. In another embodiment, n' is 2, m' is 2, and n" is 0. In another embodiment, n' is 2, m' is 3, and n" is 0.

[0545] In yet another embodiment, n' is 3, m' is 0, and n" is 0. In yet another embodiment, n' is 3, m' is 1, and n" is 0. In yet another embodiment, n' is 3, m' is 2, and n" is 0. In yet another embodiment, n' is 3, m' is 3, and n" is 0.

[0546] In a further embodiment, n' is 4, m' is 0, and n" is 0. In a further embodiment, n' is 4, m' is 1, and n" is 0. In a further embodiment, n' is 4, m' is 2, and n" is 0. In a further embodiment, n' is 4, m' is 3, and n" is 0.

[0547] In a further embodiment, n' is 5, m' is 0, and n" is 0. In a further embodiment, n' is 5, m' is 1, and n" is 0. In a further embodiment, n' is 5, m' is 2, and n" is 0. In a further embodiment, n' is 5, m' is 3, and n" is 0.

[0548] In certain embodiments, n' is 1, m' is 0, and n" is 1. In certain embodiments, n' is 1, m' is 1, and n" is 1. In certain embodiments, n' is 1, m' is 2, and n" is 1. In certain embodiments, n' is 1, m' is 3, and n" is 1.

[0549] In another embodiment, n' is 2, m' is 0, and n" is 1. In another embodiment, n' is 2, m' is 1, and n" is 1. In another embodiment, n' is 2, m' is 2, and n" is 1. In another embodiment, n' is 2, m' is 3, and n" is 1.

[0550] In yet another embodiment, n' is 3, m' is 0, and n" is 1. In yet another embodiment, n' is 3, m' is 1, and n" is 1. In yet another embodiment, n' is 3, m' is 2, and n" is 1. In yet another embodiment, n' is 3, m' is 3, and n" is 1.

[0551] In a further embodiment, n' is 4, m' is 0, and n" is 1. In a further embodiment, n' is 4, m' is 1, and n" is 1. In a further embodiment, n' is 4, m' is 2, and n" is 1. In a further embodiment, n' is 4, m' is 3, and n" is 1.

[0552] In a further embodiment, n' is 5, m' is 0, and n" is 1. In a further embodiment, n' is 5, m' is 1, and n" is 1. In a further embodiment, n' is 5, m' is 2, and n" is 1. In a further embodiment, n' is 5, m' is 3, and n" is 1.

[0553] In certain embodiments, n' is 1, m' is 0, and n" is 2. In certain embodiments, n' is 1, m' is 1, and n" is 2. In certain embodiments, n' is 1, m' is 2, and n" is 2. In certain embodiments, n' is 1, m' is 3, and n" is 2.

[0554] In another embodiment, n' is 2, m' is 0, and n" is 2. In another embodiment, n' is 2, m' is 1, and n" is 2. In another embodiment, n' is 2, m' is 2, and n" is 2. In another embodiment, n' is 2, m' is 3, and n" is 2.

[0555] In yet another embodiment, n' is 3, m' is 0, and n" is 2. In yet another embodiment, n' is 3, m' is 1, and n" is 2. In yet another embodiment, n' is 3, m' is 2, and n" is 2. In yet another embodiment, n' is 3, m' is 3, and n" is 2.

[0556] In a further embodiment, n' is 4, m' is 0, and n" is 2. In a further embodiment, n' is 4, m' is 1, and n" is 2. In a further embodiment, n' is 4, m' is 2, and n" is 2. In a further embodiment, n' is 4, m' is 3, and n" is 2.

[0557] In a further embodiment, n' is 5, m' is 0, and n" is 2. In a further embodiment, n' is 5, m' is 1, and n" is 2. In a further embodiment, n' is 5, m' is 2, and n" is 2. In a further embodiment, n' is 5, m' is 3, and n" is 2.

[0558] In certain embodiments, n' is 1, m' is 0, and n" is 3. In certain embodiments, n' is 1, m' is 1, and n" is 3. In certain embodiments, n' is 1, m' is 2, and n" is 3. In certain embodiments, n' is 1, m' is 3, and n" is 3.

[0559] In another embodiment, n' is 2, m' is 0, and n" is 3. In another embodiment, n' is 2, m' is 1, and n" is 3. In another embodiment, n' is 2, m' is 2, and n" is 3. In another embodiment, n' is 2, m' is 3, and n" is 3.

[0560] In yet another embodiment, n' is 3, m' is 0, and n" is 3. In yet another embodiment, n' is 3, m' is 1, and n" is 3. In yet another embodiment, n' is 3, m' is 2, and n" is 3. In yet another embodiment, n' is 3, m' is 3, and n" is 3.

[0561] In a further embodiment, n' is 4, m' is 0, and n" is 3. In a further embodiment, n' is 4, m' is 1, and n" is 3. In a further embodiment, n' is 4, m' is 2, and n" is 3. In a further embodiment, n' is 4, m' is 3, and n" is 3.

[0562] In a further embodiment, n' is 5, m' is 0, and n" is 3. In a further embodiment, n' is 5, m' is 1, and n" is 3. In a further embodiment, n' is 5, m' is 2, and n" is 3. In a further embodiment, n' is 5, m' is 3, and n" is 3.

[0563] In certain embodiments, n' is 1, m' is 0, and n" is 4. In certain embodiments, n' is 1, m' is 1, and n" is 4. In certain embodiments, n' is 1, m' is 2, and n" is 4. In certain embodiments, n' is 1, m' is 3, and n" is 4.

[0564] In another embodiment, n' is 2, m' is 0, and n" is 4. In another embodiment, n' is 2, m' is 1, and n" is 4. In another embodiment, n' is 2, m' is 2, and n" is 4. In another embodiment, n' is 2, m' is 3, and n" is 4.

[0565] In yet another embodiment, n' is 3, m' is 0, and n" is 4. In yet another embodiment, n' is 3, m' is 1, and n" is 4. In yet another embodiment, n' is 3, m' is 2, and n" is 4. In yet another embodiment, n' is 3, m' is 3, and n" is 4.

[0566] In a further embodiment, n' is 4, m' is 0, and n" is 4. In a further embodiment, n' is 4, m' is 1, and n" is 4. In a further embodiment, n' is 4, m' is 2, and n" is 4. In a further embodiment, n' is 4, m' is 3, and n" is 4.

[0567] In a further embodiment, n' is 5, m' is 0, and n" is 4. In a further embodiment, n' is 5, m' is 1, and n" is 4. In a further embodiment, n' is 5, m' is 2, and n" is 4. In a further embodiment, n' is 5, m' is 3, and n" is 4.

[0568] In certain embodiments, n' is 1, m' is 0, and n" is 5. In certain embodiments, n' is 1, m' is 1, and n" is 5. In certain embodiments, n' is 1, m' is 2, and n" is 5. In certain embodiments, n' is 1, m' is 3, and n" is 5.

[0569] In another embodiment, n' is 2, m' is 0, and n" is 5. In another embodiment, n' is 2, m' is 1, and n" is 5. In another embodiment, n' is 2, m' is 2, and n" is 5. In another embodiment, n' is 2, m' is 3, and n" is 5.

[0570] In yet another embodiment, n' is 3, m' is 0, and n" is 5. In yet another embodiment, n' is 3, m' is 1, and n" is 5. In yet another embodiment, n' is 3, m' is 2, and n" is 5. In yet another embodiment, n' is 3, m' is 3, and n" is 5.

[0571] In a further embodiment, n' is 4, m' is 0, and n" is 5. In a further embodiment, n' is 4, m' is 1, and n" is 5. In a further embodiment, n' is 4, m' is 2, and n" is 5. In a further embodiment, n' is 4, m' is 3, and n" is 5.

[0572] In a further embodiment, n' is 5, m' is 0, and n" is 5. In a further embodiment, n' is 5, m' is 1, and n" is 5. In a further embodiment, n' is 5, m' is 2, and n" is 5. In a further embodiment, n' is 5, m' is 3, and n" is 5.

[0573] Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0574] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is O(CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n”CH2C═O and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-10. In one embodiment, m is selected from 1-3 and n″ is selected from 0-5. In one embodiment, m is selected from 1-2 and n″ is selected from 0-3. In one embodiment, m is selected from 1-2 and n″ is selected from 0-2. In a particular embodiment, m is 1 and n″ is 0. In another embodiment, m is 2 and n″ is 0. In yet another embodiment, m is 3 and n″ is 0. In a further embodiment, m is 4 and n″ is 0. In a particular embodiment, m is 1 and n″ is 1. In another embodiment, m is 2 and n″ is 1. In yet another embodiment, m is 3 and n″ is 1. In further embodiments, m is 4 and n" is 1. In certain embodiments, m is 1 and n" is 2. In another embodiment, m is 2 and n" is 2. In yet another embodiment, m is 3 and n" is 2. In further embodiments, m is 4 and n" is 2. In certain embodiments, m is 1 and n" is 3. In another embodiment, m is 2 and n" is 3. In yet another embodiment, m is 3 and n" is 3. In further embodiments, m is 4 and n" is 3. In certain embodiments, m is 1 and n" is 4. In another embodiment, m is 2 and n" is 4. In yet another embodiment, m is 3 and n" is 4. In further embodiments, m is 4 and n" is 4. In certain embodiments, m is 1 and n" is 5. In another embodiment, m is 2 and n" is 5. In yet another embodiment, m is 3 and n" is 5. In a further embodiment, m is 4 and n" is 5. In a particular embodiment, m is 1 and n" is 6. In another embodiment, m is 2 and n" is 6. In yet another embodiment, m is 3 and n" is 6. In a further embodiment, m is 4 and n" is 6. Preferably, the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0575] In one embodiment, the present invention comprises a sugar covalently conjugated to a carrier protein (CP) through a spacer, the sugar having the general formula (VII), wherein X is O(CH2CHO) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4.

[0576] In one embodiment, m is selected from 1 to 3, m' is selected from 0 to 4, and n" is selected from 0 to 10. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n" is selected from 0 to 5. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 3. In one embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n" is selected from 0 to 1.

[0577] In certain embodiments, m is 1, m' is 0, and n" is 0. In other embodiments, m is 1, m' is 1, and n" is 0. In other embodiments, m is 1, m' is 2, and n" is 0. In other embodiments, m is 1, m' is 3, and n" is 0.

[0578] In another embodiment, m is 2, m' is 0, and n" is 0. In another embodiment, m is 2, m' is 1, and n" is 0. In another embodiment, m is 2, m' is 2, and n" is 0. In another embodiment, m is 2, m' is 3, and n" is 0.

[0579] In yet another embodiment, m is 3, m' is 0, and n" is 0. In yet another embodiment, m is 3, m' is 1, and n" is 0. In yet another embodiment, m is 3, m' is 2, and n" is 0. In yet another embodiment, m is 3, m' is 3, and n" is 0.

[0580] In a further embodiment, m is 4, m' is 0, and n" is 0. In a further embodiment, m is 4, m' is 1, and n" is 0. In a further embodiment, m is 4, m' is 2, and n" is 0. In a further embodiment, m is 4, m' is 3, and n" is 0.

[0581] In certain embodiments, m is 1, m' is 0, and n" is 1. In certain embodiments, m is 1, m' is 1, and n" is 1. In certain embodiments, m is 1, m' is 2, and n" is 1. In certain embodiments, m is 1, m' is 3, and n" is 1.

[0582] In another embodiment, m is 2, m' is 0, and n" is 1. In another embodiment, m is 2, m' is 1, and n" is 1. In another embodiment, m is 2, m' is 2, and n" is 1. In another embodiment, m is 2, m' is 3, and n" is 1.

[0583] In yet another embodiment, m is 3, m' is 0, and n" is 1. In yet another embodiment, m is 3, m' is 1, and n" is 1. In yet another embodiment, m is 3, m' is 2, and n" is 1. In yet another embodiment, m is 3, m' is 3, and n" is 1.

[0584] In a further embodiment, m is 4, m' is 0, and n" is 1. In a further embodiment, m is 4, m' is 1, and n" is 1. In a further embodiment, m is 4, m' is 2, and n" is 1. In a further embodiment, m is 4, m' is 3, and n" is 1.

[0585] In certain embodiments, m is 1, m' is 0, and n" is 2. In certain embodiments, m is 1, m' is 1, and n" is 2. In certain embodiments, m is 1, m' is 2, and n" is 2. In certain embodiments, m is 1, m' is 3, and n" is 2.

[0586] In another embodiment, m is 2, m' is 0, and n" is 2. In another embodiment, m is 2, m' is 1, and n" is 2. In another embodiment, m is 2, m' is 2, and n" is 2. In another embodiment, m is 2, m' is 3, and n" is 2.

[0587] In yet another embodiment, m is 3, m' is 0, and n" is 2. In yet another embodiment, m is 3, m' is 1, and n" is 2. In yet another embodiment, m is 3, m' is 2, and n" is 2. In yet another embodiment, m is 3, m' is 3, and n" is 2.

[0588] In a further embodiment, m is 4, m' is 0, and n" is 2. In a further embodiment, m is 4, m' is 1, and n" is 2. In a further embodiment, m is 4, m' is 2, and n" is 2. In a further embodiment, m is 4, m' is 3, and n" is 2.

[0589] In certain embodiments, m is 1, m' is 0, and n" is 3. In certain embodiments, m is 1, m' is 1, and n" is 3. In certain embodiments, m is 1, m' is 2, and n" is 3. In certain embodiments, m is 1, m' is 3, and n" is 3.

[0590] In another embodiment, m is 2, m' is 0, and n" is 3. In another embodiment, m is 2, m' is 1, and n" is 3. In another embodiment, m is 2, m' is 2, and n" is 3. In another embodiment, m is 2, m' is 3, and n" is 3.

[0591] In yet another embodiment, m is 3, m' is 0, and n" is 3. In yet another embodiment, m is 3, m' is 1, and n" is 3. In yet another embodiment, m is 3, m' is 2, and n" is 3. In yet another embodiment, m is 3, m' is 3, and n" is 3.

[0592] In a further embodiment, m is 4, m' is 0, and n" is 3. In a further embodiment, m is 4, m' is 1, and n" is 3. In a further embodiment, m is 4, m' is 2, and n" is 3. In a further embodiment, m is 4, m' is 3, and n" is 3.

[0593] In certain embodiments, m is 1, m' is 0, and n" is 4. In certain embodiments, m is 1, m' is 1, and n" is 4. In certain embodiments, m is 1, m' is 2, and n" is 4. In certain embodiments, m is 1, m' is 3, and n" is 4.

[0594] In another embodiment, m is 2, m' is 0, and n" is 4. In another embodiment, m is 2, m' is 1, and n" is 4. In another embodiment, m is 2, m' is 2, and n" is 4. In another embodiment, m is 2, m' is 3, and n" is 4.

[0595] In yet another embodiment, m is 3, m' is 0, and n" is 4. In yet another embodiment, m is 3, m' is 1, and n" is 4. In yet another embodiment, m is 3, m' is 2, and n" is 4. In yet another embodiment, m is 3, m' is 3, and n" is 4.

[0596] In a further embodiment, m is 4, m' is 0, and n" is 4. In a further embodiment, m is 4, m' is 1, and n" is 4. In a further embodiment, m is 4, m' is 2, and n" is 4. In a further embodiment, m is 4, m' is 3, and n" is 4.

[0597] In certain embodiments, m is 1, m' is 0, and n" is 5. In certain embodiments, m is 1, m' is 1, and n" is 5. In certain embodiments, m is 1, m' is 2, and n" is 5. In certain embodiments, m is 1, m' is 3, and n" is 5.

[0598] In another embodiment, m is 2, m' is 0, and n" is 5. In another embodiment, m is 2, m' is 1, and n" is 5. In another embodiment, m is 2, m' is 2, and n" is 5. In another embodiment, m is 2, m' is 3, and n" is 5.

[0599] In yet another embodiment, m is 3, m' is 0, and n" is 5. In yet another embodiment, m is 3, m' is 1, and n" is 5. In yet another embodiment, m is 3, m' is 2, and n" is 5. In yet another embodiment, m is 3, m' is 3, and n" is 5.

[0600] In a further embodiment, m is 4, m' is 0, and n" is 5. In a further embodiment, m is 4, m' is 1, and n" is 5. In a further embodiment, m is 4, m' is 2, and n" is 5. In a further embodiment, m is 4, m' is 3, and n" is 5.

[0601] Preferably the saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0602] Preferably, the saccharide of the glycoconjugates of the invention is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

[0603] 1.5 Carrier Proteins for the Glycoconjugates of the Invention One component of a glycoconjugate is a carrier protein to which the sugar is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" may be used interchangeably herein. The carrier protein should be amenable to standard conjugation procedures.

[0604] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is DT (diphtheria toxoid), TT (tetanus toxoid), or fragment C of TT, CRM 197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT mutants (e.g., CRM 176 , CRM 228 , CRM 45 (Uchida et al. (1973) J. Biol. Chem. 218:3838~3844), CRM9, CRM 102 , CRM 103 , or CRM 107and other mutations described by Nicholls and Youle, Genetically Engineered Toxins, edited by Frankel, Maecel Dekker Inc., (1992); deletion of Glu-148 or mutation to Asp, Gln, or Ser and / or deletion of Ala-158 or mutation to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; deletion of at least one or more residues Lys 516, Lys 526, Phe 530, and / or Lys 534 mutation and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711, ply detoxified in some manner, e.g., pneumococcal pneumolysin (ply) including dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formol (Kuo et al. (1995) Infect. Immun, 63:2706-2713), PhtX including PhtA, PhtB, PhtD, PhtE (the sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO00 / 37105 and WO00 / 39299), and protein fusions such as PhtDE fusions, PhtBE fusions, Pht A-E (WO01 / 98334, WO03 / 054007, WO2009 / 000826), OMPC (meningococcal outer membrane protein) usually extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (N.meningitidis), PD (Haemophilus influenzae protein D, see e.g., EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP 0 378 881, EP 0 427 347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP 0 471 177), cytokines, lymphokines, growth factors, or hormones (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes from antigens derived from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), e.g., N19 protein (Baraldoi et al. (2004) Infect Immun, 72:4884-4887), pneumococcal surface protein PspA (WO02 / 091998), iron uptake protein (WO01 / 72337), Clostridium difficile toxin A or B (WO00 / 61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa aeruginosa exotoxin A (particularly atoxic mutants thereof, such as exotoxin A with a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol., 169(11):4967-4971)). 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. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in WO 2004 / 083251), Escherichia coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa. Another suitable carrier protein is C5a peptidase (SCP) from Streptococcus.

[0605] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a TT, DT, DT mutant (CRM 197 ), or C5a peptidase (SCP) from Streptococcus.

[0606] In one embodiment, the carrier protein of the glycoconjugates of the invention is DT (diphtheria toxoid), hi another embodiment, the carrier protein of the glycoconjugates of the invention is TT (tetanus toxoid).

[0607] In another embodiment, the carrier protein of the glycoconjugates of the invention is PD (H. influenzae protein D, see for example EP0594610 B).

[0608] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a CRM 197 or C5a peptidase (SCP) from Streptococcus.

[0609] In a preferred embodiment, the carrier protein of the glycoconjugate of the present invention is a CRM 197 CRM 197 The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. 197 The nontoxigenic phage β197 was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage β. tox- CRM is produced by Corynebacterium diphtheriae infected with HIV (Uchida et al. (1971) Nature New Biology 233:8-11). 197 The protein has the same molecular weight as diphtheria toxin but differs from it by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid to glycine) in the mature protein, eliminating the toxic properties of diphtheria toxin. CRM 197Proteins are safe and effective T cell-dependent carriers for sugars. 197 and further details regarding its production can be found, for example, in US Pat. No. 5,614,382.

[0610] In one embodiment, the carrier protein of the glycoconjugates of the present invention is a CRM 197 (See CN103495161). In one embodiment, the glycoconjugate carrier protein of the present invention is a CRM obtained through expression in genetically recombinant Escherichia coli (E. coli). 197 It is the A chain of (see No. CN103495161).

[0611] In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is SCP (streptococcal C5a peptidase). Two important species of β-hemolytic streptococci, namely Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS), which cause a variety of serious human infections ranging from mild pharyngitis and impetigo to severe invasive diseases such as necrotizing fasciitis (GAS) and neonatal sepsis (GBS), have developed ways to subvert this immune response. All human isolates of β-hemolytic streptococci, including GAS and GBS, produce a highly conserved cell wall protein, SCP (streptococcal C5a peptidase), which specifically inactivates C5a. The scp genes from GAS and GBS encode polypeptides containing 1,134–1,181 amino acids (Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391–18396). The first 31 residues are the presequence of the export signal, which is removed upon passage through the cytoplasmic membrane. The next 68 residues serve as the prosequence and must be removed to generate an active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting at Lys-1034, there are four consecutive 17-residue motifs, followed by cell sorting and cell wall attachment signals. This combined signal consists of a 20-residue hydrophilic sequence containing the LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus.

[0612] SCPs can be divided into domains (see Figure 1B in Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). These domains include the pre / pro domain (which contains the export signal pre-sequence (typically the first 31 residues) and pro-sequence (typically the next 68 residues)), the protease domain (which is divided into two parts: protease part 1 is typically residues 89-333 / 334, and protease domain part 2 is typically residues 467 / 468-583 / 584), and the protease-associated domain (PA domain) (see Figure 1B in Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). These domains are typically residues 333 / 334 to 467 / 468), three fibronectin type III (Fn) domains (Fn1, typically residues 583 / 584 to 712 / 713; Fn2, typically residues 712 / 713 to 928 / 929 / 930; and Fn3, typically residues 929 / 930 to 1029 / 1030 / 1031), and a cell wall anchor domain (typically C-terminal residues 1029 / 1030 / 1031).

[0613] In one embodiment, the carrier protein of the glycoconjugate of the present invention is SCP from GBS (SCPB). An example of SCPB is provided in SEQ ID NO: 3 of WO 97 / 26008. See also SEQ ID NO: 3 of WO 00 / 34487.

[0614] In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is SCP (SCPA) from GAS. Examples of SCPA can be found in SEQ ID NO: 1 and SEQ ID NO: 2 of WO 97 / 26008. See also SEQ ID NO: 1, 2, and 23 of WO 00 / 34487.

[0615] In a preferred embodiment, the carrier protein of the glycoconjugates of the present invention is an enzymatically inactive SCP.

[0616] In another preferred embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive SCP from GBS (SCPB).

[0617] In another preferred embodiment, the carrier protein of the glycoconjugates of the present invention is an enzymatically inactive SCP (SCPA) from GAS.

[0618] In one embodiment, the carrier protein of the glycoconjugate of the present invention is a fragment of SCP. In one embodiment, the carrier protein of the glycoconjugate of the present invention is a fragment of SCPA. Preferably, the carrier protein of the glycoconjugate of the present invention is a fragment of SCPB.

[0619] In one embodiment, the glycoconjugate carrier protein of the invention is a fragment of SCP that contains the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but does not contain the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0620] In one embodiment, the glycoconjugate carrier protein of the invention is a fragment of SCP that contains the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but does not contain the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0621] In one embodiment, the glycoconjugate carrier protein of the invention is an enzymatically inactive fragment of SCP that contains the protease domain, the protease-associated domain (PA domain), and two of the three fibronectin type III (Fn) domains, but does not contain the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0622] In one embodiment, the glycoconjugate carrier protein of the invention is an enzymatically inactive fragment of an SCP, which in one embodiment contains the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not contain the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0623] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPA, which comprises the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not comprise the pre-sequence, pro-sequence, or cell wall anchor domain of the export signal.

[0624] In a preferred embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPB, which preferably contains the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not contain the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal.

[0625] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least one amino acid in the wild-type sequence. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. Numbers indicate amino acid residue positions in the peptidase according to the numbering of SEQ ID NO: 1 in WO00 / 34487.

[0626] Thus, in one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0627] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0628] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0629] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A.

[0630] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0631] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0632] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, said at least one amino acid replacement is in the protease domain. In one embodiment, said at least one amino acid replacement is in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, the substitution is S512A.

[0633] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least two amino acids of the wild-type sequence. In one embodiment, the at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the at least two amino acid replacements are D130A and H193A. In one embodiment, the at least two amino acid replacements are D130A and N295A. In one embodiment, the at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid replacements are H193A and N295A. In one embodiment, the at least two amino acid replacements are H193A and S512A. In one embodiment, the at least two amino acid replacements are N295A and S512A.

[0634] Thus, in one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, said at least two amino acid replacements are D130A and N295A. Preferably, said at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0635] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, said at least two amino acid replacements are D130A and N295A. Preferably, said at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0636] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, said at least two amino acid replacements are D130A and N295A. Preferably, said at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0637] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, said at least two amino acid replacements are D130A and N295A. Preferably, said at least two amino acid replacements are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0638] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, the at least two amino acid substitutions are D130A and N295A. Preferably, the at least two amino acid substitutions are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0639] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, the at least two amino acid substitutions are D130A and N295A. Preferably, the at least two amino acid substitutions are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0640] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, said at least two amino acid replacements are in the protease domain. In one embodiment, said at least two amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least two amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least two amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least two amino acid replacements are D130A and H193A. In one embodiment, the at least two amino acid substitutions are D130A and N295A. Preferably, the at least two amino acid substitutions are D130A and S512A. In one embodiment, the at least two amino acid substitutions are H193A and N295A. In one embodiment, the at least two amino acid substitutions are H193A and S512A. In one embodiment, the at least two amino acid substitutions are N295A and S512A.

[0641] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least three amino acids of the wild-type sequence. In one embodiment, the at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, the at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, the at least three amino acid replacements are D130A, N295A, and S512A. In one embodiment, the at least three amino acid replacements are H193A, N295A, and S512A.

[0642] Thus, in one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0643] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0644] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0645] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of an SCP, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0646] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, the at least three amino acid substitutions are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0647] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, the at least three amino acid substitutions are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0648] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, said at least three amino acid replacements are in the protease domain. In one embodiment, said at least three amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least three amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least three amino acid replacements are selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, said at least three amino acid replacements are D130A, H193A, and N295A. In one embodiment, the at least three amino acid substitutions are D130A, H193A, and S512A. In one embodiment, the at least three amino acid substitutions are D130A, N295A, and S512A. In one embodiment, the at least three amino acid substitutions are H193A, N295A, and S512A.

[0649] In one embodiment, the enzymatic activity of the SCP is inactivated by replacing at least four amino acids of the wild-type sequence, hi one embodiment, the replacement of at least four amino acids is D130A, H193A, N295A, and S512A.

[0650] Thus, in one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCP, wherein said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0651] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPA, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0652] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive SCPB, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0653] In one embodiment, the glycoconjugate carrier protein of the invention is an enzymatically inactive fragment of SCP, and said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0654] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCP comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but not the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0655] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPA comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least one amino acid replacement is in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0656] In one embodiment, the glycoconjugate carrier protein of the present invention is an enzymatically inactive fragment of SCPB comprising the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but excluding the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, wherein said inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, said at least four amino acid replacements are in the protease domain. In one embodiment, said at least four amino acid replacements are in part 1 of the protease domain. In one embodiment, said at least four amino acid replacements are in part 2 of the protease domain. In one embodiment, said at least four amino acid replacements are D130A, H193A, N295A, and S512A.

[0657] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:41.

[0658] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:42.

[0659] SEQ ID NO:41: MAKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTTKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKVVLMSSATALYDEDEK AYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

[0660] SEQ ID NO:41 is 950 amino acids in length.

[0661] SEQ ID NO:42: AKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTTKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKVVLMSSATALYDEDEK AYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

[0662] SEQ ID NO:42 is 949 amino acids in length.

[0663] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:41.

[0664] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:41.

[0665] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:41.

[0666] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity to SEQ ID NO:41.

[0667] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:41.

[0668] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:41.

[0669] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:42.

[0670] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:42.

[0671] In a particular embodiment, the carrier protein of the glycoconjugate of the invention is an enzymatically inactive fragment of an SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:42.

[0672] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity to SEQ ID NO:42.

[0673] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:42.

[0674] In a particular embodiment, the carrier protein of the glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:42.

[0675] 2 Immunogenic composition 2.1 Glycoconjugate Combinations of the Invention In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention (as disclosed in section 1 above).

[0676] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention and comprising 1 to 25 different glycoconjugates.

[0677] In one embodiment, the invention relates to an immunogenic composition comprising a glycoconjugate of the invention, said composition comprising 1 to 25 glycoconjugates (1 to 25 pneumococcal conjugates) from different serotypes of S. pneumoniae. In one embodiment, the invention relates to an immunogenic composition comprising glycoconjugates from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition comprises glycoconjugates from 16 or 20 different serotypes of S. pneumoniae. In one embodiment, the immunogenic composition is a 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 14-, 15-, 16-, 17-, 18-, or 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 16-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 19-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0678] In one embodiment, the immunogenic composition is a 21-, 22-, 23-, 24-, or 25-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition. In one embodiment, the immunogenic composition is a 25-valent pneumococcal conjugate composition.

[0679] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. In one embodiment, the immunogenic composition is an 11-valent pneumococcal conjugate composition.

[0680] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the immunogenic composition is a 13-valent pneumococcal conjugate composition.

[0681] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 15-valent pneumococcal conjugate composition.

[0682] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 20-valent pneumococcal conjugate composition.

[0683] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention and further comprising a glycoconjugate from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0684] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0685] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 2, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0686] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 2, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0687] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0688] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0689] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0690] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0691] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0692] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0693] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0694] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0695] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0696] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0697] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0698] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0699] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0700] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0701] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0702] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0703] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, and 33F. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0704] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0705] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0706] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0707] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0708] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0709] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0710] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0711] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0712] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0713] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, and 33F. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0714] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 33F, and 35B. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0715] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0716] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0717] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0718] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B. In one embodiment, the immunogenic composition is a 25-valent pneumococcal conjugate composition.

[0719] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38.

[0720] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 21-valent pneumococcal conjugate composition.

[0721] In one embodiment, the present invention relates to an immunogenic composition comprising the glycoconjugates of the present invention, wherein the glycoconjugates comprise 21 glycoconjugates selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 22-valent pneumococcal conjugate composition.

[0722] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention and comprising at least one glycoconjugate selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 19A, 19F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38.

[0723] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, said composition comprising 22 glycoconjugates selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 19A, 19F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0724] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, said composition comprising 23 glycoconjugates selected from the group consisting of glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 19A, 19F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 24-valent pneumococcal conjugate composition.

[0725] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 2, 9N, 15A, 17F, 20, 23A, 23B, 24F, and 35B. In one embodiment, the immunogenic composition is a 10-valent pneumococcal conjugate composition.

[0726] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the present invention, comprising glycoconjugates from S. pneumoniae serotypes 2, 9N, 15A, 17F, 19A, 19F, 20, 23A, 23B, 24F, and 35B. In one embodiment, the immunogenic composition is a 12-valent pneumococcal conjugate composition.

[0727] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention and comprising glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 23-valent pneumococcal conjugate composition.

[0728] In one embodiment, the present invention relates to an immunogenic composition comprising a glycoconjugate of the invention, comprising glycoconjugates from S. pneumoniae serotypes 2, 7C, 9N, 10B, 15A, 16F, 17F, 19A, 19F, 20, 21, 22A, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, and 38. In one embodiment, the immunogenic composition is a 25-valent pneumococcal conjugate composition.

[0729] In a preferred embodiment, each saccharide is individually conjugated to a different molecule of protein carrier (each molecule of protein carrier has only one type of saccharide conjugated to it). In said embodiment, the capsular saccharides are said to be individually conjugated to the carrier protein. Preferably, all glycoconjugates of the immunogenic composition are individually conjugated to the carrier protein.

[0730] In one embodiment of any of the above immunogenic compositions, the glycoconjugates of the invention comprise a CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate of the invention is conjugated to an SCP.

[0731] In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 22F is 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 33F is conjugated to a CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 15B is conjugated to a CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 12F is conjugated to a CRM 197In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 10A is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 11A is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugate from S. pneumoniae serotype 8 is conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F are conjugated to a CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 1, 5, and 7F are conjugated to CRM 197 In one embodiment of any of the above immunogenic compositions, the glycoconjugates from S. pneumoniae serotypes 6A and 19A are conjugated to a CRM 197 It is conjugated with.

[0732] In one embodiment of any of the above immunogenic compositions, the glycoconjugates of any of the above immunogenic compositions are all CRM 197 It is conjugated with.

[0733] In one embodiment of any of the above immunogenic compositions, the glycoconjugate of the invention is conjugated to an SCP and all other glycoconjugates are individually conjugated to a CRM 197 It is conjugated with.

[0734] In one embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, and / or 23F of any of the above immunogenic compositions are individually conjugated to PD.

[0735] In one embodiment, the glycoconjugate from S. pneumoniae serotype 18C of any of the above immunogenic compositions is conjugated to TT.

[0736] In one embodiment, the glycoconjugate from S. pneumoniae serotype 19F of any of the above immunogenic compositions is conjugated to DT.

[0737] In one embodiment, the glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, and / or 23F of any of the above immunogenic compositions are individually conjugated to PD, the glycoconjugate from S. pneumoniae serotype 18C is conjugated to TT, and the glycoconjugate from S. pneumoniae serotype 19F is conjugated to DT.

[0738] In one embodiment, the immunogenic composition comprises between 8 and 25 different serotypes of S. pneumoniae.

[0739] The compositions of the present invention may contain small amounts of free carrier. When a given carrier protein is present in both free and conjugated form in the compositions of the present invention, the unconjugated form preferably does not exceed 5% of the total amount of carrier protein in the entire composition, more preferably less than 2% by weight.

[0740] 2.2 Dosage of the immunogenic composition of the present invention The amount of glycoconjugate in each dose is selected to induce an immunoprotective response in a typical vaccine without significant adverse side effects, and such amount will vary depending on which specific immunogen is used and how it is presented.

[0741] The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on the total saccharide (conjugated and unconjugated) of that conjugate. For example, a glycoconjugate with 20% free saccharides will have about 80 μg of conjugated saccharides and about 20 μg of unconjugated saccharides in a 100 μg saccharide dose. The amount of glycoconjugate may vary depending on the bacterium and bacterial serotype. The saccharide concentration can be determined by uronic acid assay.

[0742] The "immunogenic amounts" of the various saccharide components in the immunogenic composition may vary, and each may contain about 0.5 μg, about 0.75 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, or about 100 μg of any particular saccharide antigen.

[0743] Generally, each dose contains between 0.1 μg and 100 μg of saccharide. In one embodiment, each dose contains between 0.1 μg and 100 μg of saccharide. In a preferred embodiment, each dose contains between 0.5 μg and 20 μg. In a preferred embodiment, each dose contains between 1.0 μg and 10 μg. In an even more preferred embodiment, each dose contains between 2.0 μg and 5.0 μg of serotype 3 polysaccharide. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0744] In one embodiment, each dose contains about 0.5 μg of sugar. In one embodiment, each dose contains about 0.55 μg of sugar. In one embodiment, each dose contains about 0.75 μg of sugar. In one embodiment, each dose contains about 1.0 μg of sugar. In one embodiment, each dose contains about 1.1 μg of sugar. In one embodiment, each dose contains about 1.5 μg of sugar. In one embodiment, each dose contains about 2.0 μg of sugar. In one embodiment, each dose contains about 2.2 μg of sugar. In one embodiment, each dose contains about 2.5 μg of sugar. In one embodiment, each dose contains about 3.0 μg of sugar. In one embodiment, each dose contains about 3.5 μg of sugar. In one embodiment, each dose contains about 4.0 μg of sugar. In one embodiment, each dose contains about 4.4 μg of sugar. In one embodiment, each dose contains about 5.0 μg of sugar. In one embodiment, each dose comprises about 5.5 μg of sugar. In one embodiment, each dose comprises about 6.0 μg of sugar.

[0745] Generally, each dose contains between 0.1 μg and 100 μg of saccharide for a given bacterium or serotype. In one embodiment, each dose contains between 0.1 μg and 100 μg of saccharide for a given bacterium or serotype. In a preferred embodiment, each dose contains between 0.5 μg and 20 μg. In a preferred embodiment, each dose contains between 1.0 μg and 10 μg. In an even more preferred embodiment, each dose contains between 2.0 μg and 5.0 μg of saccharide for a given bacterium or serotype. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0746] In one embodiment, each dose contains about 0.5 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 0.55 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 0.75 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 1.0 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 1.1 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 1.5 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 2.0 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 2.2 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 2.5 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 3.0 μg of sugar for each specific glycoconjugate. In one embodiment, each dose contains about 3.5 μg of sugar for each particular glycoconjugate. In one embodiment, each dose contains about 4.0 μg of sugar for each particular glycoconjugate. In one embodiment, each dose contains about 4.4 μg of sugar for each particular glycoconjugate. In one embodiment, each dose contains about 5.0 μg of sugar for each particular glycoconjugate. In one embodiment, each dose contains about 5.5 μg of sugar for each particular glycoconjugate. In one embodiment, each dose contains about 6.0 μg of sugar for each particular glycoconjugate.

[0747] 2.3 Carrier amount Generally, each dose comprises 10 μg to 150 μg of carrier protein, particularly 15 μg to 100 μg of carrier protein, more particularly 25 μg to 75 μg of carrier protein, and even more particularly 40 μg to 60 μg of carrier protein. In one embodiment, the carrier protein is CRM. 197 In one embodiment, the carrier protein is SCP.

[0748] In one embodiment, each dose is about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg g, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, or about 75 μg of carrier protein.

[0749] In one embodiment, each dose is about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, about 68 μg, about 69 μg, about 70 μg, about 71 μg, about 72 μg, about 73 μg, about 74 μg, or about 75 μg of carrier protein.

[0750] In one embodiment, each dose contains about 30 μg of carrier protein. In one embodiment, each dose contains about 31 μg of carrier protein. In one embodiment, each dose contains about 32 μg of carrier protein. In one embodiment, each dose contains about 33 μg of carrier protein. In one embodiment, each dose contains about 34 μg of carrier protein. In one embodiment, each dose contains about 45 μg of carrier protein.

[0751] In one embodiment, each dose contains about 40 μg of carrier protein. In one embodiment, each dose contains about 41 μg of carrier protein. In one embodiment, each dose contains about 42 μg of carrier protein. In one embodiment, each dose contains about 43 μg of carrier protein. In one embodiment, each dose contains about 44 μg of carrier protein. In one embodiment, each dose contains about 45 μg of carrier protein.

[0752] In one embodiment, each dose contains about 48 μg of carrier protein. In one embodiment, each dose contains about 49 μg of carrier protein. In one embodiment, each dose contains about 50 μg of carrier protein. In one embodiment, each dose contains about 51 μg of carrier protein. In one embodiment, each dose contains about 52 μg of carrier protein. In one embodiment, each dose contains about 53 μg of carrier protein.

[0753] In one embodiment, the carrier protein is a CRM 197 is.

[0754] In one embodiment, the carrier protein is SCP.

[0755] 2.4 Further antigens The immunogenic compositions of the present invention comprise conjugated saccharide antigens (conjugate glycoproteins). These may also further comprise antigens from other pathogens, in particular bacteria and / or viruses. Preferred further antigens are selected from diphtheria toxoid (D), tetanus toxoid (T), pertussis antigen (P) (typically acellular (Pa)), hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis A virus (HAV) antigen, conjugated Haemophilus influenzae type b capsular saccharide (Hib), inactivated poliovirus vaccine (IPV).

[0756] In one embodiment, the immunogenic composition of the invention comprises DT-Pa. In one embodiment, the immunogenic composition of the invention comprises DT-Pa-Hib, DT-Pa-IPV, or DT-Pa-HBsAg. In one embodiment, the immunogenic composition of the invention comprises DT-Pa-HBsAg-IPV or DT-Pa-HBsAg-Hib. In one embodiment, the immunogenic composition of the invention comprises DT-Pa-HBsAg-IPV-Hib.

[0757] Pertussis antigens: Bordetella pertussis causes whooping cough. Pertussis antigens in vaccines are either cellular (whole cells in the form of inactivated B. pertussis cells) or acellular. The preparation of cellular pertussis antigens is well documented (e.g., they can be obtained by heat inactivation of phase I cultures of B. pertussis). Preferably, however, the present invention uses acellular antigens. When acellular antigens are used, it is preferred to use one, two, or (preferably) three of the following antigens: (1) detoxified pertussis toxin (pertussis toxoid or PT), (2) filamentous hemagglutinin (FHA), and (3) pertactin (also known as a 69-kilodalton outer membrane protein). FHA and pertactin may be treated with formaldehyde before use according to the present invention. PT is preferably detoxified by treatment with formaldehyde and / or glutaraldehyde. The acellular pertussis antigens are preferably adsorbed onto one or more aluminum salt adjuvants. Alternatively, they can be added in an unadsorbed state. When pertactin is added, it is preferably already adsorbed onto aluminum hydroxide adjuvant. PT and FHA can be adsorbed onto aluminum hydroxide adjuvant or aluminum phosphate adjuvant. Most preferably, PT, FHA, and pertactin are all adsorbed onto aluminum hydroxide.

[0758] Inactivated Poliovirus Vaccine: Poliovirus causes poliomyelitis. Rather than using an oral poliovirus vaccine, a preferred embodiment of the present invention uses IPV. Prior to administration to a patient, the poliovirus must be inactivated, which can be achieved by treatment with formaldehyde. Poliomyelitis can be caused by one of three types of poliovirus. While the three types are similar and cause the same symptoms, they are antigenically distinct, and infection with one type does not protect against infection with the others. Therefore, it is preferred to use three poliovirus antigens in the present invention: poliovirus type 1 (e.g., Mahoney strain), poliovirus type 2 (e.g., MEF-1 strain), and poliovirus type 3 (e.g., Saukett strain). The viruses are preferably grown, purified, and inactivated individually and then combined together to provide a bulk trivalent mixture for use in the present invention.

[0759] Diphtheria toxoid: Corynebacterium diphtheriae causes diphtheria. Diphtheria toxin can be treated (e.g., with formalin or formaldehyde) to remove toxicity while retaining the ability to induce specific antitoxin antibodies after injection. These diphtheria toxoids are used in diphtheria vaccines. Preferred diphtheria toxoids are those prepared by formaldehyde treatment. Diphtheria toxoids can be obtained by growing C. diphtheriae in a growth medium, followed by formaldehyde treatment, ultrafiltration, and precipitation. The toxoided material can then be processed by processes including sterile filtration and / or dialysis. Diphtheria toxoid is preferably adsorbed onto an aluminum hydroxide adjuvant.

[0760] Tetanus toxoid: Clostridium tetani causes tetanus. Tetanus toxin can be processed to produce a protective toxoid. The toxoid is used in tetanus vaccines. A preferred tetanus toxoid is one prepared by formaldehyde treatment. Tetanus toxoid can be obtained by growing C. tetani in a growth medium, followed by formaldehyde treatment, ultrafiltration, and precipitation. The material can then be processed by processes including sterile filtration and / or dialysis.

[0761] Hepatitis A virus antigen: Hepatitis A virus (HAV) is one of the known agents that causes viral hepatitis. Preferred HAV components are based on inactivated virus, which can be achieved by formalin treatment.

[0762] Hepatitis B virus (HBV) is one of the known agents that cause viral hepatitis. The major component of the capsid is a protein known as the HBV surface antigen, or more commonly HBsAg, which is typically a 226 amino acid polypeptide with a molecular weight of approximately 24 kDa. All existing hepatitis B vaccines contain HBsAg, which, when administered to a normal recipient, stimulates the production of anti-HBsAg antibodies that protect against HBV infection.

[0763] For vaccine production, HBsAg has been produced in two ways: by purification of the antigen in particulate form from the plasma of chronic hepatitis B carriers, or by expression of the protein by recombinant DNA methods (e.g., recombinant expression in yeast cells). Unlike native HBsAg (i.e., the plasma-purified product), yeast-expressed HBsAg is generally not glycosylated, and this is the most preferred form of HBsAg for use in the present invention.

[0764] Conjugated Haemophilus influenzae type b antigens: Haemophilus influenzae type b (Hib) causes bacterial meningitis. Hib vaccines are typically based on capsular saccharide antigens, the preparation of which is well documented. Hib saccharides can be conjugated to carrier proteins to enhance their immunogenicity, especially in children. Typical carrier proteins are tetanus toxoid, diphtheria toxoid, CRM 197 , H. influenzae protein D, and an outer membrane protein complex from serogroup B Neisseria meningitidis. The sugar moiety of the conjugate may comprise full-length polyribosylribitol phosphate (PRP) prepared from Hib bacteria and / or fragments of full-length PRP. The Hib conjugate may or may not be adsorbed to an aluminum salt adjuvant.

[0765] 2.5 Adjuvants In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two, or three adjuvants. In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise two adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An antigen may act primarily as a delivery system, act primarily as an immunomodulator, or possess strong properties of both.

[0766] Suitable adjuvants include those suitable for use in mammals, including humans. Examples of known suitable delivery system-type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween™ 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0767] In one embodiment, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In a preferred embodiment, the immunogenic compositions disclosed herein comprise aluminum phosphate as an adjuvant.

[0768] Additional exemplary adjuvants for enhancing the effectiveness of the immunogenic compositions disclosed herein include, but are not limite...

Claims

1. (a) reacting the isolated capsular saccharide with a carbonate derivative and an azido linker in an aprotic solvent to generate an activated azido saccharide; (b) reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, where the NHS moiety reacts with the amino group to form an amide bond, thereby yielding an alkyne-functionalized carrier protein; (c) Cu ＋１ reacting the activated azido sugar of step (a) with the activated alkyne carrier protein of step (b) via a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate; 2. A method for making a capsular glycoconjugate comprising:

2. 2. The method of claim 1, wherein the isolated capsular saccharide is sized to a weight average molecular weight of from 200 kDa to 800 kDa prior to the activation step (a).

3. 3. The method according to any one of claims 1 to 2, wherein the carbonic acid derivative is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.

4. The azide linker is represented by the formula (I): 【Chemistry 1】 [Wherein, X is CH ２ (CH ２ ) ｎ , (CH ２ CH ２ O) ｍ CH ２ CH ２ , NHCO(CH ２ ) ｎ , NHCO(CH ２ CH ２ O) ｍ CH ２ CH ２ , O.C.H. ２ (CH ２ ) ｎ , and O(CH ２ CH ２ O) ｍ CH ２ CH ２ wherein n is selected from 1 to 10 and m is selected from 1 to 4.

5. The azide linker has the formula (II): 【Chemistry 2】 The method according to any one of claims 1 to 3, wherein the compound is

6. 6. The method of any one of claims 1 to 5, wherein the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

7. 6. The method of any one of claims 1 to 5, wherein the drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a drug having an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne.

8. The drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has the formula (III): 【Chemistry 3】 [Wherein, X is CH ２ O (CH ２ ) ｎ CH ２ C=O and CH ２ O (CH ２ CH ２ O) ｍ (CH ２ ) ｎ CH ２ C═O, n is selected from 0 to 10, and m is selected from 0 to 4. The method according to any one of claims 1 to 5, wherein the compound is

9. The drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has the formula (IV): 【Chemistry 4】 The method according to any one of claims 1 to 5, wherein the compound is

10. 10. A method according to any one of claims 1 to 9, wherein step a) comprises reacting the capsular saccharide with a carbonate derivative, followed by reacting the carbonate-activated capsular saccharide with an azido linker in an aprotic solvent to generate an activated azido capsular saccharide.

11. 11. The method of any one of claims 1 to 10, wherein in step a) the isolated capsular saccharide is reacted with CDI in an aprotic solvent comprising 0.1% to 1% (v / v) water.

12. 12. The method according to any one of claims 1 to 11, wherein in step a) activation of the carbonic acid derivative is followed by addition of water.

13. 13. The method of any one of claims 1 to 12, wherein step a) further comprises reacting the carbonic acid derivative-activated sugar with an amount of azide linker that is 0.01 to 10 molar equivalents relative to the amount of sugar repeating units of the activated sugar.

14. 14. The method according to any one of claims 1 to 13, wherein the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst.

15. 15. The method of any one of claims 1 to 14, further comprising the step of purifying the glycoconjugate after it has been produced.

16. 16. A capsular glycoconjugate produced by the method of any one of claims 1 to 15.

17. , which comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VII): 【Chemistry 5】 [Wherein, X is CH ２ (CH ２ ) ｎ’ , (CH ２ CH ２ O) ｍ CH ２ CH ２ , NHCO(CH ２ ) ｎ’ , NHCO(CH ２ CH ２ O) ｍ CH ２ CH ２ , O.C.H. ２ (CH ２ ) ｎ’ , and O(CH ２ CH ２ O) ｍ CH ２ CH ２ wherein n' is selected from 1 to 10 and m is selected from 1 to 4; X' is CH ２ O (CH ２ ) ｎ” CH ２ C=O, CH ２ O (CH ２ CH ２ O) ｍ’ (CH ２ ) ｎ” CH ２ C=O, n″ is selected from 0-10, and m′ is selected from 0-4.

18. and comprising a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer, and having the general formula (VII), ２ (CH ２ ) ｎ’ where n' is 2 and X' is CH ２ O (CH ２ ) ｎ” CH ２ C═O and n″ is 1.

19. The capsular saccharide comprises a capsular saccharide covalently conjugated to a carrier protein (CP) via a spacer and has the general formula (VIII): 【Chemistry 6】 Capsular glycoconjugates having the following structure:

20. 20. A capsular glycoconjugate according to any one of claims 16 to 19, comprising a capsular saccharide, wherein the capsular saccharide has a weight average molecular weight (Mw) prior to conjugation of between 10 kDa and 2,000 kDa.

21. 21. A capsular glycoconjugate according to any one of claims 16 to 20, having a weight average molecular weight (Mw) of from 250 kDa to 20,000 kDa.

22. 22. The capsular glycoconjugate according to any one of claims 16 to 21, wherein the degree of conjugation of the capsular glycoconjugate is between 2 and 15.

23. 23. Capsular glycoconjugate according to any one of claims 16 to 22, wherein the ratio of capsular saccharide to carrier protein (w / w) in the glycoconjugate is between 0.5 and 3.

0.

24. 24. The capsular glycoconjugate according to any one of claims 16 to 23, wherein the capsular glycoconjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 5 to 10 saccharide repeating units of the saccharide.

25. The carrier protein is a CRM １９７ 25. A capsular glycoconjugate according to any one of claims 16 to 24 which is SCP, DT (diphtheria toxoid) TT, (tetanus toxoid) or PD (H. influenzae protein D).

26. 26. A method for making a capsular glycoconjugate according to any one of claims 1 to 15, or a capsular glycoconjugate according to any one of claims 16 to 25, wherein the capsular saccharide is a capsular saccharide from a pathogenic bacterium.

27. 26. A method for making a capsular glycoconjugate according to any one of claims 1 to 15, or a capsular glycoconjugate according to any one of claims 16 to 25, wherein the capsular saccharide is a capsular saccharide from S. pneumoniae.

28. 28. A method of making a capsular glycoconjugate or a capsular glycoconjugate according to any one of claims 1 to 27, wherein the capsular saccharide is not a capsular saccharide from Streptococcus pneumoniae serotype 3.

29. 29. An immunogenic composition comprising a capsular glycoconjugate according to any one of claims 16 to 28.

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