Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Abstract

To provide immunogenic compositions which are able to generate a more robust immune response to Streptococcus pneumoniae serotype 3.SOLUTION: An immunogenic composition comprises a Streptococcus pneumoniae serotype 3 glycoconjugate produced by a method comprising the steps of: (a) reacting a Streptococcus pneumoniae serotype 3 capsular polysaccharide with a carbonic acid derivative and an azido linker in an aprotic solvent to produce an activated azido polysaccharide; (b) reacting a carrier protein with an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group, where the NHS moiety reacts with the amino groups to form an amide linkage thereby obtaining an alkyne functionalized carrier protein; and (c) reacting the activated azido polysaccharide of the step (a) with the activated alkyne carrier protein of the step (b) by Cu+1 mediated azide-alkyne cycloaddition reaction to form a glycoconjugate.SELECTED DRAWING: Figure 2

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 serotype of Streptococcus pneumoniae. The invention also relates to the vaccination of human subjects, particularly infants and the elderly, against pneumococcal infection using said glycoconjugates. [Background technology]

[0002] 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 immunized with free polysaccharide (Anderson et al. (1985) J. Pediatr. 107:346; Insel et al. (1986) J. Exp. Med. 158:294).

[0003] 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.

[0004] 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.

[0005] Pneumococcal polysaccharides, particularly capsular polysaccharides, are important immunogens found on the surface of the bacterium. This has made them important components in the design of pneumococcal vaccines. They have proven useful in eliciting immune responses, especially when coupled to carrier proteins.

[0006] Some serotypes, particularly Streptococcus pneumoniae serotype 3, produce large, viscous polysaccharide chains (e.g., glucose / glucuronic acid chains of 2–3 million daltons for type 3), which makes them difficult to handle.

[0007] Furthermore, significant immunogenicity has been difficult to achieve with serotype 3 polysaccharide. For example, in an immunogenicity and safety study of the 11-valent pneumococcal protein D conjugate vaccine (11-Pn-PD), no priming effect was observed for serotype 3 in infants who received three doses of the vaccine followed by a booster dose of either the same vaccine or a pneumococcal polysaccharide vaccine (Nurkka et al. (2004) Ped. Inf. Dis. J., 23:1008-1014). In another study, opsonophagocytosis assay (OPA) results from infants who received a dose of 11-Pn-PD failed to demonstrate antibody responses to serotype 3 at levels comparable to other tested serotypes (Gatchalian et al., 17th Annual Meeting of the Eur. Soc. Paed. Inf. Dis. (ESPID), Poster No. 4, PIA Poster Session 1, Istanbul, Turkey, March 27, 2001). In yet another study evaluating the effectiveness of 11-Pn-PD in preventing acute otitis media, the vaccine did not protect against episodes caused by serotype 3 (Prymula et al., The Lancet, Vol. 367:740-748 (March 4, 2006)). Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, there is a need for antigens that can generate a more robust immune response against Streptococcus pneumoniae serotype 3. [Means for solving the problem]

[0009] The present invention specifically provides Streptococcus pneumoniae serotype 3 glycoconjugates that exhibit improved immunogenicity. The present invention also provides processes that result in improved conjugation yields of Streptococcus pneumoniae serotype 3 glycoconjugates.

[0010] Summary of the Invention In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) reacting isolated Streptococcus pneumoniae serotype 3 capsular polysaccharide with a carbonic acid derivative and an azide linker in an aprotic solvent to produce an activated azido polysaccharide; (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 polysaccharide 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 for producing Streptococcus pneumoniae serotype 3 glycoconjugates, comprising:

[0011] In certain embodiments, the isolated polysaccharide is sized to a weight average molecular weight of between 100 kDa and 200 kDa prior to the activation step (a).

[0012] In one embodiment, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI).

[0013] In one aspect, the invention relates to Streptococcus pneumoniae serotype 3 glycoconjugates produced according to the above method.

[0014] In one aspect, the present invention comprises a Streptococcus pneumoniae serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, and having the general formula (VII):

[0015] [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 0 to 10, and m' is selected from 0 to 4. The present invention relates to Streptococcus pneumoniae serotype 3 glycoconjugates having the following structure:

[0016] In a further aspect, the present invention relates to an immunogenic composition comprising said Streptococcus pneumoniae serotype 3 glycoconjugates. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the repeating polysaccharide structure of S. pneumoniae serotype 3 capsular polysaccharide. [Figure 2]Figure 1 shows a general scheme for the preparation of Streptococcus pneumoniae serotype 3 glycoconjugates of the invention prepared using click chemistry. Pn3 poly = S. pneumoniae serotype 3 capsular polysaccharide, CP = carrier protein, CDI = 1,1'-carbonyldiimidazole. [Figure 3] Figure 1 shows the opsonophagocytic activity (OPA) titers of serotype 3-CRM197 conjugates in mice containing polysaccharides of various sizes. Mice were vaccinated with sized serotype 3 polysaccharides (approximately 25, 150, or 250 kDa) conjugated to CRM197 using either RAC / aqueous or RAC / DMSO conjugation. [Figure 4] Figure 1 shows the opsonophagocytic activity (OPA) titers of serotype 3-CRM197 conjugates in mice with different degrees of activation (DoA). Mice were vaccinated with sized serotype 3 polysaccharide conjugated to CRM197 using either RAC / aqueous or RAC / DMSO conjugation. [Figure 5] Figure 1 shows the opsonophagocytic activity (OPA) titers of serotype 3 conjugates with CRM197, SCP, or tetanus toxoid (TT) in mice using reductive amination in DMSO (RAC / DMSO). [Figure 6] Figure 1 shows the opsonophagocytic activity (OPA) titers of serotype 3-CRM197 conjugates in mice using various chemistries: reductive amination in aqueous (RAC / aqueous), reductive amination in DMSO (RAC / DMSO), eTEC-linked glycoconjugates (eTEC), or click chemistry (click). [Figure 7]Figure 1 shows OPA titers after dose 1 in rhesus macaque infants vaccinated with serotype 3 chemistry / carrier conjugates. Different chemistries were used (reductive amination in aqueous (RAC / aqueous) or click chemistry (click). Opsonophagocytic titers were measured from serum collected at 4 weeks after dose 1 across different conjugation chemistries. Each point represents an individual animal, and data are presented as geometric mean titers with 95% confidence intervals. Statistical significance was determined based on one-way ANOVA. Tukey's multiplicity-adjusted p-values ​​are reported. ****=p≦0.0001, LLOQ—lower limit of quantification. [Figure 8] Figure 1 shows OPA responses after dose 2 in infant rhesus macaques vaccinated with serotype 3 chemistry / carrier conjugates. Opsonophagocytic titers were measured from serum collected 4 weeks after dose 2 across different conjugation chemistries. Each point represents an individual animal and data are presented as geometric mean titers with 95% confidence intervals. Statistical significance was determined based on one-way ANOVA. Tukey's multiplicity-adjusted p-values ​​are reported. LLOQ - Lower Limit of Quantitation DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. The glycoconjugate of the present invention The present invention is directed, in part, to conjugated capsular saccharide antigens (also called glycoconjugates), where the saccharide is derived from a serotype of S. pneumoniae, particularly serotype 3. For the purposes of the present invention, the term "glycoconjugate" refers to a capsular saccharide covalently linked to a carrier protein. In one embodiment, the capsular saccharide is directly linked to the carrier protein. In a second embodiment, the bacterial saccharide is linked to the protein via a spacer / linker.

[0019] 1.1 Pneumococcal glycoproteins from S. pneumoniae serotype 3 As shown in Figure 1, the polysaccharide repeating unit of serotype 3 consists of a linear disaccharide unit with one glucopyranose (Glcp) and one glucuronic acid (GlcpA) (see, e.g., Geno K et al. (2015) Clin Microbiol Rev 28:3, 871-899).

[0020] In one embodiment, the encapsulated S. pneumoniae serotype 3 saccharide for use in the present invention is a synthetic carbohydrate. Preparation of synthetic Streptococcus pneumoniae type 3 capsular saccharide can be carried out, for example, as disclosed in WO2017178664.

[0021] However, in a preferred embodiment, the source of bacterial polysaccharides according to the present invention may be bacterial cells of Streptococcus pneumoniae serotype 3. Bacterial strains that can be used as a source of Streptococcus pneumoniae serotype 3 polysaccharides may be obtained from established culture collections (such as the Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA, USA)) or from clinical specimens.

[0022] Serotype 3 polysaccharides 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. They can also be purchased commercially (e.g., from the American Type Culture Collection (ATCC), Manassas, Virginia, USA (e.g., reference numbers ATCC172-X or ATCC33-X)).

[0023] When serotype 3 polysaccharide is obtained directly from bacteria, bacterial cells can be grown in a medium, preferably a soy-based medium. Fermentation of the bacterial cells that produce S. pneumoniae serotype 3 capsular polysaccharide can be followed by lysing the bacterial cells to produce a cell lysate, which can then be purified by centrifugation, depth filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration, and / or Serotype 3 polysaccharide can be isolated from the cell lysate using purification techniques known in the art, including the use of column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, and WO2008 / 118752). The purified serotype 3 capsular polysaccharide can then be used to prepare immunogenic conjugates.

[0024] The isolated serotype 3 capsular polysaccharide obtained by purification of the serotype 3 polysaccharide from S. pneumoniae lysate and optional sizing of the purified polysaccharide can be characterized by various parameters including, for example, the weight average molecular weight (Mw).

[0025] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0026] In a preferred embodiment, the isolated serotype 3 capsular polysaccharide (i.e., purified prior to further processing) has a weight average molecular weight of 5 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 5 kDa to 100 kDa.

[0027] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 50 kDa to 100 kDa.

[0028] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 100 kDa to 200 kDa.

[0029] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 150 kDa to 200 kDa.

[0030] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 200 kDa to 300 kDa.

[0031] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 300 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of between 300 kDa and 400 kDa.

[0032] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 400 kDa to 500 kDa.

[0033] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 500 kDa to 1000 kDa.

[0034] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 1500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 750 kDa to 1000 kDa.

[0035] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1000 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1000 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1000 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1000 kDa to 2000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1000 kDa to 1500 kDa.

[0036] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1500 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1500 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1500 kDa to 3000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 1500 kDa to 2000 kDa.

[0037] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2000 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2000 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2000 kDa to 3000 kDa.

[0038] In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2500 kDa to 5000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2500 kDa to 4000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide has a weight average molecular weight of 2500 kDa to 3000 kDa.

[0039] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0040] Preferably, to generate serotype 3 conjugates with advantageous filterability characteristics, immunogenicity, and / or yield, polysaccharide sizing to a target molecular weight range is performed prior to conjugation with a carrier protein. Advantageously, the purified serotype 3 polysaccharide is reduced in size while preserving critical features of the polysaccharide structure. Mechanical or chemical sizing may be used. In one embodiment, the purified serotype 3 polysaccharide is reduced in size by chemical hydrolysis. Chemical hydrolysis may be performed using a mild acid (e.g., acetic acid, formic acid, propanoic acid). In one embodiment, chemical hydrolysis is performed using formic acid. In one embodiment, chemical hydrolysis is performed using propanoic acid. In a preferred embodiment, chemical hydrolysis is performed using acetic acid. Chemical hydrolysis may also be performed 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 performed using dilute hydrochloric acid. In one embodiment, chemical hydrolysis is performed using dilute sulfuric acid. In one embodiment, chemical hydrolysis is performed 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. The size of the purified serotype 3 polysaccharide can also be reduced by mechanical homogenization. In one embodiment, the size of the purified serotype 3 polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves high shear rates by pumping the process stream through a channel with sufficiently small dimensions. The shear rate can be increased by using a higher homogenization pressure, and the exposure time can be increased by recirculating the feed stream through the homogenizer.

[0041] A high-pressure homogenization process may be suitable to reduce the size of the purified serotype 3 polysaccharide while preserving the structural features of the polysaccharide.

[0042] In a preferred embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 250 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 5 kDa and 100 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 5 kDa and 50 kDa.

[0043] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 250 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 50 kDa to 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 50 kDa and 100 kDa.

[0044] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 250 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 100 kDa and 150 kDa.

[0045] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 110 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 120 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 110 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 130 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 120 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 120 kDa to 140 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 120 kDa to 130 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 130 kDa to 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 130 kDa to 140 kDa.

[0046] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 250 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 150 kDa to 200 kDa.

[0047] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 200 kDa to 250 kDa.

[0048] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 250 kDa to 300 kDa.

[0049] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 300 kDa to 350 kDa.

[0050] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 350 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 350 kDa and 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 350 kDa and 400 kDa.

[0051] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 400 kDa to 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 400 kDa and 450 kDa.

[0052] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 600 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 450 kDa to 500 kDa.

[0053] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 600 kDa.

[0054] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 500 kDa to 600 kDa.

[0055] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 600 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 600 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 600 kDa to 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 600 kDa to 700 kDa.

[0056] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 700 kDa and 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 700 kDa and 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 700 kDa and 800 kDa.

[0057] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 800 kDa and 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 800 kDa and 900 kDa.

[0058] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of between 900 kDa and 1000 kDa.

[0059] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 5 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 50 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 100 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 110 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 120 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 130 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 140 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 150 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 160 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 170 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 180 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 190 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 200 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 250 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 300 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 350 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 400 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 450 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 500 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 550 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 600 kDa.In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of about 1000 kDa.

[0060] In one embodiment, the isolated serotype 3 capsular polysaccharide is not sized.

[0061] As described further below, the isolated serotype 3 capsular polysaccharides described above may be activated (e.g., chemically activated) to allow them to react (e.g., with a linker or directly with a carrier protein) and then be incorporated into glycoconjugates.

[0062] For purposes of the present invention, the term "glycoconjugate" refers to a sugar covalently linked to a carrier protein. In one embodiment, the sugar is directly linked to the carrier protein. In a second embodiment, the sugar is linked to the carrier protein through a spacer / linker.

[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 Streptococcus pneumoniae serotype 3 glycoconjugates of the present invention In some embodiments, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein said polysaccharide has a weight average molecular weight (Mw) of between 10 kDa and 2,000 kDa prior to conjugation.

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

[0066] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 50 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 50 kDa and 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 140 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 130 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 120 kDa. In one embodiment, the weight average molecular weight (Mw) is 50 kDa to 110 kDa.

[0067] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 75 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 75 kDa and 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 140 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 130 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 120 kDa. In one embodiment, the weight average molecular weight (Mw) is 75 kDa to 110 kDa.

[0068] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 100 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 100 kDa and 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 150 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 140 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 130 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 120 kDa. In one embodiment, the weight average molecular weight (Mw) is 100 kDa to 110 kDa.

[0069] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 125 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 125 kDa and 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 150 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 140 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 130 kDa.

[0070] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 130 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is between 130 kDa and 200 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 150 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 140 kDa.

[0071] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is 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 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 600 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 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 150 kDa and 300 kDa. In one embodiment, the weight average molecular weight (Mw) is from 150 kDa to 200 kDa.

[0072] In one embodiment, the serotype 3 glycoconjugates of the present invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is 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 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 600 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 400 kDa. In one embodiment, the weight average molecular weight (Mw) is between 200 kDa and 300 kDa.

[0073] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 300 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 300 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 300 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 300 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 300 kDa and 500 kDa. In one embodiment, the weight average molecular weight (Mw) is between 300 kDa and 400 kDa.

[0074] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is between 400 kDa and 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is between 400 kDa and 750 kDa. In one embodiment, the weight average molecular weight (Mw) is between 400 kDa and 700 kDa. In one embodiment, the weight average molecular weight (Mw) is between 400 kDa and 600 kDa. In one embodiment, the weight average molecular weight (Mw) is between 400 kDa and 500 kDa.

[0075] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein the polysaccharide has a weight average molecular weight (Mw) of 500 kDa to 1,000 kDa prior to conjugation. In one embodiment, the weight average molecular weight (Mw) is 500 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 500 kDa to 700 kDa. In one embodiment, the weight average molecular weight (Mw) is 500 kDa to 600 kDa.

[0076] In one embodiment, a serotype 3 glycoconjugate of the invention comprises a serotype 3 capsular polysaccharide, wherein the polysaccharide has a weight average molecular weight (Mw) of 600 kDa to 1,000 kDa prior to conjugation. In one embodiment, the weight average molecular weight (Mw) is 600 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 600 kDa to 700 kDa.

[0077] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein said polysaccharide has a weight average molecular weight (Mw) of 700 kDa to 1,000 kDa prior to conjugation, hi one embodiment, the weight average molecular weight (Mw) is 700 kDa to 750 kDa.

[0078] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein said polysaccharide has a weight average molecular weight (Mw) of between 750 kDa and 1,000 kDa prior to conjugation.

[0079] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0080] In one embodiment, the serotype 3 glycoconjugates of the invention comprise a serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide prior to conjugation is about 1,000 kDa. 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 140 kDa. In one embodiment, the weight average molecular weight (Mw) is about 130 kDa. In one embodiment, the weight average molecular weight (Mw) is about 120 kDa. In one embodiment, the weight average molecular weight (Mw) is about 110 kDa. In one embodiment, the weight average molecular weight (Mw) is about 100 kDa.

[0081] In some such embodiments, serotype 3 glycoconjugates are prepared using click chemistry (see section 1.3).

[0082] In some embodiments, the serotype 3 glycoconjugates of the invention have a weight average molecular weight (Mw) of 250 kDa to 20,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 15,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 10,000 kDa.

[0083] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 5,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 4,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 3,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 2,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 1,500 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 1,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 750 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 600 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 500 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 250 kDa to 400 kDa.

[0084] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 5,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 4,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 3,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 2,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 1,500 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 1,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 750 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 500 kDa to 600 kDa.

[0085] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 5,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 4,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 3,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 2,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 1,500 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 750 kDa to 1,000 kDa.

[0086] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 5,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 4,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 3,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 2,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 1,000 kDa to 1,500 kDa.

[0087] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 5,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 4,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 3,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 2,000 kDa to 3,500 kDa.

[0088] In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of between 2,250 kDa and 3,500 kDa.

[0089] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 3,000 kDa to 5,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of between 3,000 kDa and 4,000 kDa.

[0090] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 4,000 kDa to 5,000 kDa.

[0091] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 5,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 5,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 5,000 kDa to 8,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 5,000 kDa to 7,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 5,000 kDa to 6,000 kDa.

[0092] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 6,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 6,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 6,000 kDa to 8,000 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 6,000 kDa to 7,000 kDa.

[0093] In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 7,000 kDa to 10,000 kDa. In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 7,000 kDa to 9,000 kDa. In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 7,000 kDa to 8,000 kDa.

[0094] In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 8,000 kDa to 10,000 kDa. In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 8,000 kDa to 9,000 kDa.

[0095] In another embodiment, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of 9,000 kDa to 10,000 kDa.

[0096] Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0097] In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 10,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 9,000 kDa. In other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 6,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 5,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 4,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 3,500 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 3,250 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 3,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 2,500 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 2,250 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 2,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 1,000 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 750 kDa. In still other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 600 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 500 kDa. In yet other embodiments, the serotype 3 glycoconjugates have a weight average molecular weight (Mw) of about 400 kDa.

[0098] The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0099] Another method for characterizing the serotype 3 glycoconjugates of the invention is to characterize them by binding to a carrier protein (e.g., CRMP). 197The degree of conjugation of a serotype 3 glycoconjugate of the present invention depends on the number of lysine residues in the carrier protein (or SCP) that are conjugated to a saccharide, which can be characterized as the extent of conjugated lysines (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent attachment to a polysaccharide 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 serotype 3 glycoconjugate of the present invention is 2 to 15. In one embodiment, the degree of conjugation of a serotype 3 glycoconjugate of the present invention is 2 to 13. In one embodiment, the degree of conjugation of a serotype 3 glycoconjugate of the present invention is 2 to 10. In one embodiment, the degree of conjugation of a serotype 3 glycoconjugate of the present invention is 2 to 8. In one embodiment, the degree of conjugation of a serotype 3 glycoconjugate of the present invention is 2 to 6. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 2 to 5. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 2 to 4. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 15. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 13. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 10. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 8. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 6. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 5. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 3 to 4. In one embodiment, the conjugation degree of the serotype 3 glycoconjugates of the present invention is 5 to 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is between 5 and 10. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is between 8 and 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is between 8 and 12.In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the invention is between 10 and 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the invention is between 10 and 12.

[0100] In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the invention is about 2.

[0101] In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 3. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 4. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 5. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 6. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 7. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 8. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 9. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 10, about 11. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 12. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is about 13. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the invention is about 14. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the invention is about 15. In a preferred embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the 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.

[0102] The serotype 3 glycoconjugates of the present invention may also be characterized by the sugar-to-carrier protein ratio (w / w). In some embodiments, the serotype 3 polysaccharide-to-carrier protein ratio (w / w) in the glycoconjugate is 0.5-3.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-2.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.8-1.2. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 0.5-1.0. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0-1.5. In other embodiments, the sugar-to-carrier protein ratio (w / w) is 1.0-2.0. In a further embodiment, the sugar-to-carrier protein ratio (w / w) is 0.8-1.2. In a preferred embodiment, the ratio of serotype 3 capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1.

[0103] 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.

[0104] Serotype 3 glycoconjugates of the invention may also be characterized by the number of covalent bonds between the carrier protein and the saccharide as a function of the repeating saccharide unit. In one embodiment, a serotype 3 glycoconjugate of the invention comprises at least one covalent bond between the carrier protein and the polysaccharide for every four saccharide repeating units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeating units of the polysaccharide. In another embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeating units of the polysaccharide. In a further embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 25 saccharide repeating units of the polysaccharide. In a further embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 50 saccharide repeating units of the polysaccharide. In a further embodiment, a covalent bond between the carrier protein and the polysaccharide occurs at least once for every 100 saccharide repeating units of the polysaccharide.

[0105] In another embodiment, the serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 5-10 saccharide repeat units of the polysaccharide.

[0106] In other embodiments, serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 2 to 7 saccharide repeat units of the polysaccharide.

[0107] In other embodiments, the serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 6 to 11 saccharide repeat units of the polysaccharide.

[0108] In other embodiments, serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 9-14 saccharide repeat units of the polysaccharide.

[0109] In another embodiment, the serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 10-20 saccharide repeat units of the polysaccharide.

[0110] In another embodiment, the serotype 3 glycoconjugates of the invention comprise at least one covalent bond between the carrier protein and the polysaccharide for every 4 to 25 saccharide repeat units of the polysaccharide.

[0111] In frequent embodiments, the carrier protein is CRM197. In frequent embodiments, the carrier protein is SCP.

[0112] In some embodiments, the carrier protein is a CRM 197 and CRM 197 and the polysaccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeating units of the polysaccharide. In frequent embodiments, the carrier protein is an SCP and the covalent bond between the SCP and the polysaccharide occurs at least once for every 4, 10, 15, or 25 saccharide repeating units of the polysaccharide.

[0113] The serotype 3 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).

[0114] In a preferred embodiment, the serotype 3 glycoconjugates comprise less than about 50% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In a preferred embodiment, the serotype 3 glycoconjugates comprise less than about 40% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In a further preferred embodiment, the serotype 3 glycoconjugates comprise less than about 25% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In a further preferred embodiment, the serotype 3 glycoconjugates comprise less than about 20% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide. In a further preferred embodiment, the serotype 3 glycoconjugates comprise less than about 15% free serotype 3 polysaccharide relative to the total amount of serotype 3 polysaccharide.

[0115] Serotype 3 glycoconjugates also exhibit a 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.

[0116] In a preferred embodiment, at least 30% of the serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 40% of the glycoconjugates 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 serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, at least 60% of the serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 50% to 80% of serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d In a preferred embodiment, 65% to 80% of serotype 3 glycoconjugates have a K of 0.3 or less in a CL-4B column. d It has.

[0117] 1.3 Streptococcus pneumoniae serotype 3 glycoconjugates of the invention prepared using click chemistry In one embodiment, the serotype 3 glycoconjugates of the invention are prepared using click chemistry.

[0118] The present invention also relates to methods of making serotype 3 glycoconjugates as disclosed herein.

[0119] According to the present invention, click chemistry involves three steps: (a) reacting isolated serotype 3 capsular polysaccharide with a carbonic acid derivative and an azide linker in an aprotic solvent to produce an activated azidopolysaccharide (polysaccharide 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 reacting the activated azido polysaccharide of step (a) with the activated alkyne carrier protein of step (b) via a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate.

[0120] After step (a), the polysaccharide is said to be activated and is referred to herein as an "activated polysaccharide" or an "activated azidopolysaccharide."

[0121] After step (b), the support is said to be activated and is called an "activated support."

[0122] As mentioned above, activation (a) can be preceded by sizing of the polysaccharide to a target molecular weight (MW) range.

[0123] Thus, in one embodiment, the isolated polysaccharide is sized prior to activation with a carbonate derivative and an azide linker.

[0124] In one embodiment, the isolated polysaccharide is sized to any of the target molecular weight (MW) ranges defined above.

[0125] 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.

[0126] In one embodiment, the carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI). In one 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.

[0127] 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).

[0128] In one embodiment, the azide linker has formula (I):

[0129] [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.

[0130] In one embodiment, the azido linker has formula (I), wherein X is CH2(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.

[0131] 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.

[0132] In one embodiment, the azido linker has formula (I), where 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] In one embodiment, the azido linker has formula (II):

[0137] [ka] is a compound of

[0138] In one embodiment, the azido linker is 3-azido-propylamine.

[0139] 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.

[0140] 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.

[0141] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has formula (III):

[0142] [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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group has formula (IV):

[0156] [ka] is a compound of

[0157] In one embodiment, step a) comprises reacting a polysaccharide with a carbonic acid derivative, followed by reacting the carbonic acid derivative-activated polysaccharide with an azide linker in an aprotic solvent to produce an activated azido polysaccharide.

[0158] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0159] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0160] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0161] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0162] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0163] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0164] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.5 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0165] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.8 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0166] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 1 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0167] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 2 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0168] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 3 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0169] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 5 to 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0170] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0171] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0172] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0173] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0174] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0175] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0176] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.5 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0177] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.8 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0178] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 1 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0179] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 2 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0180] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 3 to 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0181] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0182] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0183] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0184] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0185] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0186] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0187] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.5 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0188] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.8 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0189] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 1 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0190] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 2 to 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0191] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0192] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0193] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0194] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0195] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0196] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0197] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.5 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0198] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.8 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0199] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 1 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0200] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0201] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0202] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0203] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0204] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0205] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0206] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.5 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0207] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.8 to 1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0208] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0209] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0210] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0211] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0212] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0213] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.4 to 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0214] In one embodiment, step a) comprises reacting the polysaccharide with an amount of a carbonic acid derivative.

[0215] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0216] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0217] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0218] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0219] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.3 to 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0220] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.01 to 0.3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0221] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.05 to 0.3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0222] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.1 to 0.3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0223] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is 0.2 to 0.3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0224] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.01 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0225] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.05 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0226] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.08 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0227] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.1 molar equivalent relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0228] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0229] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0230] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0231] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 0.5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0232] In one embodiment, step a) comprises reacting the polysaccharide with about 1 molar equivalent of the carbonic acid derivative relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0233] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0234] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 3 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0235] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 4 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0236] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 5 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0237] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 8 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0238] In one embodiment, step a) comprises reacting the polysaccharide with an amount of carbonic acid derivative that is about 10 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture.

[0239] In one embodiment, in step a), the isolated polysaccharide is reacted with a carbonic acid derivative in an aprotic solvent.

[0240] In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of dimethyl formamide (DMF). In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0241] In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of dimethylacetamide. In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of N-methyl-2-pyrrolidone. In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in a solution consisting essentially of hexamethylphosphoramide (HMPA).

[0242] In a preferred embodiment, the isolated polysaccharide is reacted with a carbonic acid derivative in a solution consisting essentially of dimethyl sulfoxide (DMSO).

[0243] In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in dimethyl formamide (DMF). In one embodiment, the isolated polysaccharide is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO).

[0244] In one embodiment, the isolated polysaccharide is reacted with a carbonic acid derivative in dimethylacetamide. In one embodiment, the isolated polysaccharide is reacted with a carbonic acid derivative in N-methyl-2-pyrrolidone. In one embodiment, the isolated polysaccharide is reacted with a carbonic acid derivative in hexamethylphosphoramide (HMPA).

[0245] In a preferred embodiment, the isolated polysaccharide is reacted with CDI in dimethyl sulfoxide (DMSO). In one embodiment, the isolated polysaccharide is reacted with CDI in anhydrous DMSO.

[0246] Surprisingly, it has been found that side reactions can be avoided by reacting the isolated polysaccharide with CDI in an environment having a moisture level of about 0.1% to 1% (v / v). Thus, in one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 0.3% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.1% to 0.2% (v / v) water.

[0247] In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.2% to 1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.2% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.2% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.2% to 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.2% to 0.3% (v / v) water.

[0248] In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.3% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.3% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing 0.3% to 0.4% (v / v) water.

[0249] In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.2% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.3% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.6% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.7% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in an aprotic solvent containing about 0.9% (v / v) water.

[0250] In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 0.3% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% to 0.2% (v / v) water.

[0251] In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.2% to 1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.2% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.2% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.2% to 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.2% to 0.3% (v / v) water.

[0252] In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% to 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% to 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% to 0.4% (v / v) water.

[0253] In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.2% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.3% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.6% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.7% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.8% (v / v) water, hi one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.9% (v / v) water.

[0254] 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.

[0255] 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).

[0256] After reacting the polysaccharide with a carbonate derivative and final quenching of the carbonate derivative with water, the carbonate-activated polysaccharide is reacted with an azide linker.

[0257] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0258] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.01 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0259] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.01 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0260] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.01 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0261] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0262] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.01 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0263] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.01 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0264] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0265] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0266] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0267] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0268] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0269] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0270] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0271] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0272] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0273] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.05 to 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0274] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0275] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0276] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0277] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0278] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0279] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.1 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0280] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0281] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.1 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0282] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0283] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0284] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0285] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0286] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0287] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide 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 polysaccharide.

[0288] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0289] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0290] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0291] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0292] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0293] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0294] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0295] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 1 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0296] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 2 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0297] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 2 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0298] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 2 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0299] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 2 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0300] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 2 to 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0301] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 3 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0302] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 3 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0303] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 3 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0304] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 3 to 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0305] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 4 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0306] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 4 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0307] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 4 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0308] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 5 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0309] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 5 to 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0310] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is 8 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0311] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 0.01 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0312] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 0.05 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0313] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 0.1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0314] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0315] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0316] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0317] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 3 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0318] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 4 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0319] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0320] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 8 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0321] In one embodiment, step a) further comprises reacting the carbonic acid derivative-activated polysaccharide with an amount of azide linker that is about 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0322] In the above embodiment, the carbonate derivative is preferably CDI. In another embodiment, the carbonate derivative is CDT.

[0323] In one embodiment, the degree of activation of the activated polysaccharide after step a) is 0.5-50%. The degree of activation of the azido polysaccharide is defined as the percentage of repeating units linked with an azido linker.

[0324] In one embodiment, the degree of activation of the activated polysaccharide after step a) is 1-30%. In another embodiment, the degree of activation of the activated polysaccharide after step a) is 2-25%. In another embodiment, the degree of activation of the activated polysaccharide after step a) is 3-20%.

[0325] In another embodiment, the degree of activation of the activated polysaccharide after step a) is 3-15%. In another embodiment, the degree of activation of the activated polysaccharide after step a) is 4-15%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is 1-6%.

[0326] In one embodiment, the degree of activation of the activated polysaccharide after step a) is 3-6%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is 10-15%.

[0327] In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 1%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 2%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 3%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 4%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 5%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 6%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 7%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 8%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 9%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 10%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 11%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 12%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 13%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 14%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 15%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 16%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 17%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 18%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 19%. In one embodiment, the degree of activation of the activated polysaccharide after step a) is about 20%.

[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 0.1 to 10 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 0.5 to 10 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 1 to 10 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 1.5 to 10 molar equivalents relative to the lysines on the carrier.

[0332] 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 10 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 2.5 to 10 molar equivalents relative to the lysines on the carrier.

[0334] 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.

[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 5 to 10 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 7.5 to 10 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 7.5 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 7.5 molar equivalents relative to the lysines on the carrier.

[0339] In one embodiment, step b) comprises reacting the carrier protein with an amount of 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.

[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 7.5 molar equivalents relative to the lysines on the carrier.

[0341] In one embodiment, step b) comprises reacting the carrier protein with an amount of 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.

[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 2.5 to 7.5 molar equivalents relative to the lysines on the carrier.

[0343] 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.

[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 5 to 7.5 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 0.1 to 5 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 0.5 to 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 1 to 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 1.5 to 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 2 to 5 molar equivalents 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 2.5 to 5 molar equivalents 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 3 to 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 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] 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.

[0373] 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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 carrier (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.

[0385] In one embodiment, the carrier protein is an SCP or a fragment thereof. In said embodiment, the activation degree of the activated carrier after step b) may be 1-50.

[0386] In another embodiment, the activation degree of the activating support (SCP) after step b) is 5 to 50. In another embodiment, the activation degree of the activating support (SCP) after step b) is 7 to 45. In another embodiment, the activation degree of the activating support (SCP) after step b) is 5 to 15. In another embodiment, the activation degree of the activating support (SCP) after step b) is 20 to 30. In another embodiment, the activation degree of the activating support (SCP) after step b) is 30 to 50. In another embodiment, the activation degree of the activating support (SCP) after step b) is 30 to 40. In another embodiment, the activation degree of the activating support (SCP) after step b) is 10 to 40. In another embodiment, the activation degree of the activating support (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 activation degree 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.

[0387] In one embodiment, the carrier protein is TT or a fragment thereof. In said embodiment, the activation degree of the activated carrier after step b) may be 1-30.

[0388] In another embodiment, the activation degree of the activating support (TT) after step b) is 5 to 25. In another embodiment, the activation degree of the activating support (TT) after step b) is 7 to 25. In another embodiment, the activation degree of the activating support (TT) after step b) is 10 to 20. In another embodiment, the activation degree of the activating support (TT) after step b) is about 5. In another embodiment, the activation degree of the activating support (TT) after step b) is about 7. In another embodiment, the activation degree of the activating support (TT) after step b) is about 10. In another embodiment, the activation degree of the activating support (TT) after step b) is about 12. In another embodiment, the activation degree of the activating support (TT) after step b) is about 15. In another embodiment, the activation degree of the activating support (TT) after step b) is about 20. In another embodiment, the activation degree 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.

[0389] 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.

[0390] In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide 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 polysaccharide 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 polysaccharide 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 polysaccharide 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 polysaccharide to activated alkyne support in step c) is about 0.5. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 0.6. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 0.7. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 0.8. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 0.9. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.1. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.2. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.3. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.4. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.5. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.6.In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.7. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.8. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 1.9. In one embodiment, the initial input ratio (weight to weight) of activated azido polysaccharide to activated alkyne support in step c) is about 2.

[0391] 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.

[0392] In one embodiment, the azide group capping agent has formula (V):

[0393] [ka] [Wherein X is (CH2) n and n is selected from 1 to 15.

[0394] In one embodiment, the azide group capping agent is propargyl alcohol.

[0395] 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.

[0396] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0397] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0398] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0399] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0400] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0401] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0402] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is 1 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

[0403] In one embodiment, the unreacted azide groups are capped 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 polysaccharide.

[0404] In one embodiment, the unreacted azide groups are capped using an amount of capping agent that is about 1 molar equivalent to the amount of polysaccharide repeat units of the activated polysaccharide.

[0405] In one embodiment, the unreacted azide groups are capped using an amount of capping agent that is about 1.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

[0406] In one embodiment, capping of unreacted azide groups is carried out with an amount of capping agent that is about 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0407] In one embodiment, capping of unreacted azide groups is carried out with an amount of capping agent that is about 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0408] 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.

[0409] In one embodiment, the alkyne group capping agent has formula (VI):

[0410] [ka] [Wherein X is (CH2) n and n is selected from 1 to 15.

[0411] In one embodiment, the alkyne group capping agent is 3-azido-1-propanol.

[0412] 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.

[0413] 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 polysaccharide.

[0414] 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 repeat units of the activated polysaccharide.

[0415] 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 repeat units of the activated polysaccharide.

[0416] 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 repeat units of the activated polysaccharide.

[0417] 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 polysaccharide.

[0418] 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 polysaccharide.

[0419] 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 polysaccharide.

[0420] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0421] 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 repeat units of the activated polysaccharide.

[0422] In one embodiment, capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0423] In one embodiment, the capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 1 molar equivalent to the amount of polysaccharide repeat units of the activated polysaccharide.

[0424] In one embodiment, the capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 1.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0425] In one embodiment, capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0426] In one embodiment, capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 2.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0427] In one embodiment, capping of unreacted alkyne groups is carried out with an amount of capping agent that is about 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated polysaccharide.

[0428] 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.

[0429] In one aspect, the present invention provides serotype 3 glycoconjugates produced according to any of the methods disclosed herein.

[0430] In one aspect, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide having the general formula (VII):

[0431] [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 0 to 10, and m' is selected from 0 to 4. The present invention provides a serotype 3 glycoconjugate having the formula:

[0432] Formula (VII) is a schematic representation of a serotype 3 glycoconjugate of the present invention. It should not be understood that linkages are present in all repeating saccharide units. Rather, the majority of the S. pneumoniae serotype 3 saccharide repeating units are left unmodified, and covalent linkages between the carrier protein and the saccharide are present in a minority of the saccharide repeating units. Furthermore, a single carrier protein (CP) molecule may be linked to multiple S. pneumoniae serotype 3 saccharide molecules, and a single S. pneumoniae serotype 3 saccharide molecule can be linked to more than one carrier protein (CP) molecule.

[0433] In a preferred embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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].

[0434] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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 certain embodiments, 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 certain embodiments, 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.

[0435] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0436] 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.

[0437] 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.

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] 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.

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] 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.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] 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.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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.

[0467] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0468] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 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.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] 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.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] 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.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] 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.

[0490] 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.

[0491] 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.

[0492] 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.

[0493] 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.

[0494] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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 certain embodiments, 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 certain embodiments, 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.

[0495] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0496] 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.

[0497] 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.

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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.

[0502] 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.

[0503] 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.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] 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.

[0508] 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.

[0509] 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.

[0510] 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.

[0511] 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.

[0512] 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.

[0513] 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.

[0514] 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.

[0515] 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.

[0516] 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.

[0517] 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.

[0518] 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.

[0519] 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.

[0520] 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.

[0521] 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.

[0522] 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.

[0523] 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.

[0524] 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.

[0525] 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.

[0526] 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.

[0527] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0528] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0529] 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.

[0530] 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.

[0531] 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.

[0532] 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.

[0533] 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.

[0534] 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.

[0535] 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.

[0536] 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.

[0537] 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.

[0538] 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.

[0539] 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.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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.

[0544] 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.

[0545] 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.

[0546] 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.

[0547] 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.

[0548] 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.

[0549] 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.

[0550] 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.

[0551] 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.

[0552] 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.

[0553] 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.

[0554] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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 certain embodiments, 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 certain embodiments, 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.

[0555] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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.

[0556] 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.

[0557] 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.

[0558] 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.

[0559] 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.

[0560] 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.

[0561] 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.

[0562] 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.

[0563] 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.

[0564] 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.

[0565] 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.

[0566] 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.

[0567] 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.

[0568] 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.

[0569] 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.

[0570] 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.

[0571] 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.

[0572] 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.

[0573] 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.

[0574] 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.

[0575] 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.

[0576] 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.

[0577] 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.

[0578] 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.

[0579] 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.

[0580] 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.

[0581] 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.

[0582] 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.

[0583] 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.

[0584] 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.

[0585] 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.

[0586] 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.

[0587] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide 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 to 10.

[0588] In one embodiment, m is selected from 1 to 3 and n" is selected from 0 to 10. In one embodiment, m is selected from 1 to 3 and n" is selected from 0 to 5. In one embodiment, m is selected from 1 to 2 and n" is selected from 0 to 3. In one embodiment, m is selected from 1 to 2 and n" is selected from 0 to 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 a further embodiment, m is 4 and n" is 1. In a particular embodiment, 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 a further embodiment, m is 4 and n" is 2. In a particular embodiment, 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 a further embodiment, m is 4 and n" is 3. In a particular embodiment, 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 a further embodiment, m is 4 and n" is 4. In a particular embodiment, 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.

[0589] In one embodiment, the present invention comprises a serotype trisaccharide covalently conjugated to a carrier protein (CP) through a spacer, the serotype trisaccharide having the general formula (VII), wherein X is O(CH2CHO) mCH2CH2, 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.

[0590] 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.

[0591] 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.

[0592] 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.

[0593] 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.

[0594] 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.

[0595] 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.

[0596] 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.

[0597] 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.

[0598] 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.

[0599] 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.

[0600] 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.

[0601] 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.

[0602] 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.

[0603] 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.

[0604] 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.

[0605] 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.

[0606] 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.

[0607] 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.

[0608] 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.

[0609] 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.

[0610] 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.

[0611] 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.

[0612] 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.

[0613] 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.

[0614] 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.

[0615] 1.6 Carrier Proteins of Streptococcus pneumoniae Serotype 3 Glycoconjugates of the Invention One component of a glycoconjugate is a carrier protein to which a purified polysaccharide 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.

[0616] In a preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 way, for example, pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect. J. Med. Soc. 1999; 1999; 2000). 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.

[0617] In a preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is selected from the group consisting of TT, DT, DT mutant (CRM 197 and C5a peptidase (SCP) from Streptococcus.

[0618] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate is DT (diphtheria toxoid). In another embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate is TT (tetanus toxoid).

[0619] In another embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate is PD (H. influenzae protein D, see for example EP0594610 B).

[0620] In a preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate is CRM 197 or C5a peptidase (SCP) from Streptococcus.

[0621] In a preferred embodiment, the serotype 3 capsular polysaccharide is CRM 197 Conjugated to a protein. 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. CRM197 Proteins 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.

[0622] In one embodiment, the serotype 3 capsular polysaccharide is CRM 197 In one embodiment, the serotype 3 capsular polysaccharide is conjugated to a CRM protein. 197 Protein or CRM 197 (See CN103495161). In one embodiment, the serotype 3 capsular polysaccharide is conjugated to the A chain of CRM obtained via expression in genetically recombinant E. coli. 197 It is conjugated to the A chain of (see CN103495161).

[0623] In another preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is SCP (Streptococcal C5a peptidase).

[0624] Two important species of β-hemolytic streptococci, 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.

[0625] 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).

[0626] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide 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.

[0627] In another preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is SCP (SCPA) from GAS.

[0628] Examples of SCPAs can be found in WO 97 / 26008 at SEQ ID NO: 1 and SEQ ID NO: 2. See also SEQ ID NOs: 1, 2, and 23 in WO 00 / 34487.

[0629] In a preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugates of the present invention is an enzymatically inactive SCP.

[0630] In another preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is enzymatically inactive SCP (SCPB) from GBS.

[0631] In another preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive SCP (SCPA) from GAS.

[0632] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugates of the invention is a fragment of SCP. In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugates of the invention is a fragment of SCPA. Preferably, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugates of the invention is a fragment of SCPB.

[0633] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate 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.

[0634] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate 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.

[0635] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate 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.

[0636] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of an SCP, which in one embodiment comprises the protease domain, the protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0637] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCPA, which in one embodiment comprises the protease domain, the protease-associated domain (PA domain), and the three fibronectin type III (Fn) domains, but does not include the export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0638] In a preferred embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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 export signal pre-sequence, pro-sequence, and cell wall anchor domain.

[0639] 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.

[0640] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive 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.

[0641] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0642] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0643] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0644] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of an 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 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, the substitution is N295A, hi yet another embodiment, the substitution is S512A.

[0645] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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, the substitution is N295A, hi yet another embodiment, the substitution is S512A.

[0646] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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, the substitution is N295A, hi yet another embodiment, the substitution is S512A.

[0647] 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.

[0648] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive 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.

[0649] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0650] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0651] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0652] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of an 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 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.

[0653] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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.

[0654] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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.

[0655] 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.

[0656] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive 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.

[0657] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0658] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0659] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0660] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of an 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 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.

[0661] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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.

[0662] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate 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 not 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.

[0663] 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.

[0664] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive 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.

[0665] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0666] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the 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.

[0667] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate 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.

[0668] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of an 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.

[0669] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the 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 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 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.

[0670] In one embodiment, the carrier protein of a serotype 3 capsular polysaccharide glycoconjugate of the 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 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.

[0671] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:41.

[0672] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO:42.

[0673] SEQ ID NO:41: MAKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTTKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKVVLMSSATALYDEDEK AYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

[0674] SEQ ID NO:41 is 950 amino acids in length.

[0675] SEQ ID NO:42: AKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTTKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKVVLMSSATALYDEDEK AYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

[0676] SEQ ID NO:42 is 949 amino acids in length.

[0677] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:41.

[0678] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:41.

[0679] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:41.

[0680] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide 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.

[0681] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:41.

[0682] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:41.

[0683] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity to SEQ ID NO:42.

[0684] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity to SEQ ID NO:42.

[0685] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99% identity to SEQ ID NO:42.

[0686] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide 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:42.

[0687] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity to SEQ ID NO:42.

[0688] In a specific embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity to SEQ ID NO:42.

[0689] 2 Immunogenic composition 2.1 Glycoconjugate Combinations of the Invention In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention.

[0690] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the invention, wherein the composition comprises from 1 to 25 different glycoconjugates.

[0691] In one embodiment, the invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the invention, 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.

[0692] 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.

[0693] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

[0694] In one embodiment, the immunogenic composition further comprises glycoconjugates from S. pneumoniae serotypes 1, 5, and 7F.

[0695] In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates from S. pneumoniae serotypes 6A and 19A.

[0696] In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates from S. pneumoniae serotypes 22F and 33F.

[0697] In one embodiment, any of the above immunogenic compositions further comprises glycoconjugates from S. pneumoniae serotypes 8, 10A, 11A, 12F, and 15B.

[0698] In one embodiment, any of the above immunogenic compositions further comprises a glycoconjugate from S. pneumoniae serotype 2.

[0699] In one embodiment, any of the above immunogenic compositions further comprises a glycoconjugate from S. pneumoniae serotype 9N.

[0700] In one embodiment, any of the above immunogenic compositions further comprises a glycoconjugate from S. pneumoniae serotype 17F.

[0701] In one embodiment, any of the above immunogenic compositions further comprises a glycoconjugate from S. pneumoniae serotype 20.

[0702] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. In one embodiment, the immunogenic composition is an octavalent pneumococcal conjugate composition.

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

[0704] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 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.

[0705] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 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.

[0706] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 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.

[0707] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes 1, 2, 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.

[0708] In one embodiment, the present invention relates to an immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate of the present invention and further comprising glycoconjugates from S. pneumoniae serotypes...

Claims

1. It comprises a Streptococcus pneumoniae serotype 3 polysaccharide covalently conjugated to a carrier protein (CP) through a spacer and has the general formula (VII): 【Transformation 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 , OCH 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 to 10, and m′ is selected from 0 to 4.

1. An immunogenic composition comprising a Streptococcus pneumoniae serotype 3 glycoconjugate having the following structure:

2. and a Streptococcus pneumoniae serotype trisaccharide covalently conjugated to a carrier protein (CP) through 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].

3. 3. The immunogenic composition of claim 1 or 2, comprising serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of said polysaccharide before conjugation is between 75 kDa and 200 kDa.

4. The immunogenic composition of claim 1 or 2, wherein the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 1,000 kDa to 4,000 kDa.

5. The carrier protein is a CRM １９７ The immunogenic composition of claim 1 or 2,

6. The immunogenic composition of claim 1 or 2, wherein the carrier protein is an SCP.

7. The immunogenic composition of claim 1 or 2, comprising 1 to 25 glycoconjugates from different serotypes of S. pneumoniae.

8. 3. The immunogenic composition of claim 1 or 2, further comprising glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, and is a 20-valent pneumococcal conjugate composition.

9. 3. The immunogenic composition of claim 1 or 2, further comprising glycoconjugates from S. pneumoniae serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B, and is a 25-valent pneumococcal conjugate composition.

Images