Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof

Abstract

The present invention relates to novel conjugated capsular saccharide antigens (glycoconjugates), immunogenic compositions comprising said glycoconjugates, and uses thereof. [Figure 1] TIFF2024522395000037.tif136127

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] Specifically, the present invention provides a Streptococcus pneumoniae serotype 3 complex carbohydrate that exhibits improved immunogenicity. The present invention also provides a process (method of preparation) for producing a Streptococcus pneumoniae serotype 3 complex carbohydrate with improved conjugation yields.

[0010] Summary of the Invention In one aspect, the present invention relates to (a) reacting an isolated Streptococcus pneumoniae serotype 3 capsular polysaccharide with a carbonate derivative and an azide linker in a non-protic solvent to produce an activated azide polysaccharide; (b) reacting a carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, wherein the NHS moiety reacts with an amino group to form an amide bond, thereby obtaining an alkyne-functionalized carrier protein; (c) reacting the activated azide polysaccharide of step (a) with the activated alkyne carrier protein of step (b) by a Cu +1 -mediated azide-alkyne cycloaddition reaction to form a complex carbohydrate. The present invention also relates to a method for producing a Streptococcus pneumoniae serotype 3 complex carbohydrate, comprising the steps of:

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

[0012] In one aspect, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI).

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

[0014] In one aspect, the present invention includes a Streptococcus pneumoniae serotype 3 sugar conjugated in common with a carrier protein (CP) through a spacer, and has the general formula (VII):

[0015] [Chemical formula] [wherein X is selected from the group consisting of 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; m is selected from 1 to 4; X' is selected from the group consisting of CH2O(CH2) n” CH2C=O, CH2O(CH2CH2O) m’ (CH2) n” CH2C=O; n'' is selected from 0 to 10; m' is selected from 0 to 4] and relates to a Streptococcus pneumoniae serotype 3 complex carbohydrate having the same.

[0016] In a further aspect, the present invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 3 complex carbohydrate.

Brief Description of the Drawings

[0017] [Figure 1] It is a diagram showing the repeating polysaccharide structure of the Streptococcus pneumoniae (S. pneumoniae) serotype 3 capsular polysaccharide. [Diagram 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 PREFERRED EMBODIMENTS

[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 having one glucopyranose (Glcp) and one glucuronic acid (GlcpA) (see, for example, Geno K et al. (2015) Clin Microbiol Rev Vol. 28:3, pages 871-899).

[0020] In one embodiment, the Streptococcus pneumoniae (S. pneumoniae) serotype 3 sugar used in the present invention is a synthetic carbohydrate. The preparation of synthetic Streptococcus pneumoniae type 3 capsular sugar can be carried out, for example, as disclosed in WO2017178664.

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

[0022] The serotype 3 polysaccharide 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, as well as WO2008 / 118752). They can also be generated using synthetic protocols known to those skilled in the art. They can also be purchased (for example, from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) (for example, reference numbers ATCC172-X or ATCC33-X)).

[0023] When obtaining serotype 3 polysaccharide directly from bacteria, the bacterial cells can be grown in a medium, preferably a soybean-based medium. After the fermentation of bacterial cells that produce Streptococcus pneumoniae (S. pneumoniae) serotype 3 capsular polysaccharide, the bacterial cells can be lysed to produce a cell lysate. Then, centrifugation, depth filtration, precipitation, ultrafiltration, treatment with activated carbon, diafiltration, and / or using purification techniques known in the art, including the use of column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381, and WO2008 / 118752), the serotype 3 polysaccharide can be isolated from the cell lysate. Subsequently, the purified serotype 3 capsular polysaccharide can be used in the preparation of immunogenic conjugates.

[0024] The isolated serotype 3 capsular polysaccharide obtained by purification of serotype 3 polysaccharide from Streptococcus pneumoniae (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 the polysaccharide can be measured by size exclusion chromatography (SEC) combined 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 300 kDa to 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 5 kDa to 100 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 5 kDa to 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 400 kDa to 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 700 kDa to 1000 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 700 kDa to 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 700 kDa to 800 kDa.

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

[0058] In one embodiment, the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 900 kDa to 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 sizes up to a weight average molecular weight of about 700 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide sizes up to a weight average molecular weight of about 800 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide sizes up to a weight average molecular weight of about 900 kDa. In one embodiment, the isolated serotype 3 capsular polysaccharide sizes up to a weight average molecular weight of about 1000 kDa.

[0060] In one embodiment, the isolated serotype 3 capsular polysaccharide does not size.

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

[0062] For the 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] Generally, the covalent conjugation of sugars to carriers enhances the immunogenicity of the sugars to convert them from T-independent antigens to T-dependent antigens and thus enable priming for immunological memory. Conjugation is particularly useful for pediatric vaccines.

[0064] 1.2 Streptococcus pneumoniae Serotype 3 Glycoconjugate of the Present Invention In some embodiments, the serotype 3 glycoconjugate of the present invention comprises a serotype 3 capsular polysaccharide, and the weight average molecular weight (Mw) of the polysaccharide before conjugation is 10 kDa to 2,000 kDa.

[0065] The weight average molecular weight (Mw) of a saccharide prior to conjugation refers to the Mw prior to activation of the 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 conjugate carbohydrate of the present invention contains serotype 3 capsular polysaccharide, and the weight average molecular weight (Mw) of the polysaccharide before conjugation is 125 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 700 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 600 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 400 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 300 kDa. In one embodiment, the weight average molecular weight (Mw) is 125 kDa to 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 conjugate carbohydrate of the present invention contains serotype 3 capsular polysaccharide, and the weight average molecular weight (Mw) of the polysaccharide before conjugation is 130 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 700 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 600 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 400 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 300 kDa. In one embodiment, the weight average molecular weight (Mw) is 130 kDa to 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 complex carbohydrate of the present invention contains serotype 3 capsular polysaccharide, and the weight average molecular weight (Mw) of the polysaccharide before conjugation is 150 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 700 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 600 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 400 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 300 kDa. In one embodiment, the weight average molecular weight (Mw) is 150 kDa to 200 kDa.

[0072] In one embodiment, the serotype 3 complex carbohydrate of the present invention contains serotype 3 capsular polysaccharide, and the weight average molecular weight (Mw) of the polysaccharide before conjugation is 200 kDa to 1,000 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 750 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 700 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 600 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 500 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 400 kDa. In one embodiment, the weight average molecular weight (Mw) is 200 kDa to 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 complex carbohydrate of the present invention has a weight average molecular weight (Mw) of 250 kDa to 20,000 kDa. In other embodiments, the serotype 3 complex carbohydrate has a weight average molecular weight (Mw) of 500 kDa to 15,000 kDa. In still other embodiments, the serotype 3 complex carbohydrate has a weight average molecular weight (Mw) of 500 kDa to 10,000 kDa.

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

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

[0088] In yet other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 2,250 kDa to 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 glycoconjugate has a weight average molecular weight (Mw) of 5,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 5,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 5,000 kDa to 8,000 kDa. In still other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 5,000 kDa to 7,000 kDa. In still other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 5,000 kDa to 6,000 kDa.

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

[0093] In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 7,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 7,000 kDa to 9,000 kDa. In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 7,000 kDa to 8,000 kDa.

[0094] In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 8,000 kDa to 10,000 kDa. In other embodiments, the serotype 3 glycoconjugate has a weight average molecular weight (Mw) of 8,000 kDa to 9,000 kDa.

[0095] In other embodiments, the serotype 3 glycoconjugate has 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 one embodiment of the present disclosure.

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

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

[0099] Another method for characterizing the serotype 3 glycoconjugate of the present invention is a carrier protein (e.g., CRM 197or SCP), and is based on the number of lysine residues conjugated to the sugar, which can be characterized as the range of conjugated lysine (degree of conjugation). Evidence of lysine modification of the carrier protein due to covalent bonding with the polysaccharide can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a decrease in the number of lysine residues recovered compared to the carrier protein starting material used to prepare the conjugate material. In a preferred embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 13. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 10. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 8. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 6. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 5. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 2 to 4. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 13. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 10. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 8. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 6. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 5. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 3 to 4. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 5 to 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 5 to 10. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 8 to 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugate of the present invention is from 8 to 12.In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present invention is between 10 and 15. In one embodiment, the degree of conjugation of the serotype 3 glycoconjugates of the present 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] The serotype 3 glycoconjugate of the present invention can also be characterized by the number of covalent bonds between the carrier protein and the sugar as a function of the sugar repeating unit. In one embodiment, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every four sugar repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every ten sugar repeating units of the polysaccharide. In another embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every fifteen sugar repeating units of the polysaccharide. In a further embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every twenty-five sugar repeating units of the polysaccharide. In a further embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every fifty sugar repeating units of the polysaccharide. In a further embodiment, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every one hundred sugar repeating units of the polysaccharide.

[0105] In other embodiments, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every 5 to 10 sugar repeating units of the polysaccharide.

[0106] In other embodiments, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every 2 to 7 sugar repeating units of the polysaccharide.

[0107] In other embodiments, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every 6 to 11 sugar repeating units of the polysaccharide.

[0108] In other embodiments, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every 9 to 14 sugar repeating units of the polysaccharide.

[0109] In other embodiments, the serotype 3 glycoconjugate of the present invention contains at least one covalent bond between the carrier protein and the polysaccharide for every 10 to 20 sugar repeating 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 and 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 azido 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 azide linker is of 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 azide linker is of 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 azide linker is of 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 azide linker is of formula (II)

[0137]

Chemical formula

[0138] In one embodiment, the azide 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 is of formula (III):

[0142] [wherein, X is selected from the group consisting of CH2O(CH2) CH2C=O and CH2O(CH2CH2O) n CH2C=O and CH2O(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 is of formula (III) [wherein, X is CH2O(CH2) nIt is a compound of CH2C=O, and n is selected from 0 to 10. In one embodiment, n is selected from 0 to 5. In one embodiment, n is selected from 0 to 4. In one embodiment, n is selected from 0 to 3. In one embodiment, n is selected from 0 to 2. In a specific embodiment, n is 0. In a specific 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 CH2O(CH2CH2O) m (CH2) n It is a compound of CH2C=O, where n is selected from 0 to 10 and m is selected from 0 to 4. In one embodiment, n is selected from 0 to 5. In one embodiment, n is selected from 0 to 4. In one embodiment, n is selected from 0 to 3. In one embodiment, n is selected from 0 to 2. In a specific embodiment, n is 0. In a specific 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. In one embodiment, m is selected from 0 to 3. In one embodiment, m is selected from 0 to 2. In a specific embodiment, m is 1. In a specific embodiment, 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 the formula (IV)

[0156]

Chemical formula

[0157] In one embodiment, step a) comprises reacting a polysaccharide with a carbonate derivative, and subsequently reacting the polysaccharide activated with the carbonate derivative with an azide linker in an aprotic solvent to produce an activated azide polysaccharide.

[0158] In one embodiment, step a) comprises reacting a polysaccharide with an amount of carbonate 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 a polysaccharide with an amount of carbonate 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 a polysaccharide with an amount of carbonate 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 between 2 and 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 a carbonate 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 a carbonate 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 a carbonate derivative that is 0.5 to 2 molar equivalents relative to the amount of serotype 3 capsular polysaccharide present in the reaction mixture. C

[0198] In one embodiment, step a) comprises reacting the polysaccharide with an amount of a carbonate 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 a carbonate 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 a carbonate 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 a carbonate 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 a carbonate 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 a carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate derivative that is 0.01 to 0.5 molar equivalent 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 the carbonate derivative that is 0.05 to 0.5 molar equivalent 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 the carbonate derivative that is 0.1 to 0.5 molar equivalent 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 the carbonate derivative that is 0.2 to 0.5 molar equivalent 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 a carbonate 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 a carbonate 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 a carbonate 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 a carbonate 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 a carbonate derivative that is about 0.05 molar equivalent 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 a carbonate derivative that is about 0.08 molar equivalent 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 a carbonate 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 a carbonate derivative that is about 0.2 molar equivalent 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 the carbonate 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 the carbonate 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 the carbonate 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 an amount of the carbonate derivative that is about 1 molar equivalent 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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 the carbonate 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% - 1% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% - 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% - 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% - 0.4% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% - 0.3% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.1% - 0.2% (v / v) water.

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

[0252] In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% - 0.8% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% - 0.5% (v / v) water. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing 0.3% - 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. In one embodiment, the isolated polysaccharide is reacted with CDI in DMSO containing about 0.9% (v / v) water.

[0254] In one embodiment, the free carbonate derivative is then quenched by adding water prior to adding the azide linker. Water can inactivate free CDI.

[0255] Thus, in one embodiment, the addition of water continues for the activation of the carbonic acid derivative. 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 the carbonic acid derivative and finally quenching the carbonic acid derivative with water, the polysaccharide activated with the carbonic acid derivative is reacted with an azide linker.

[0257] In one embodiment, step a) further comprises reacting the polysaccharide activated with the carbonic acid derivative with an amount of azide linker that is 0.01 to 10 molar equivalents relative to the amount (molar equivalent of RU) of the polysaccharide repeating unit of the activated polysaccharide.

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

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

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

[0261] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of an azide linker that is 0.01 to 3 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

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

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

[0264] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of an azide linker that is 0.01 to 0.5 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0265] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of an azide linker that is 0.01 to 0.1 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0266] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of an azide linker that is 0.05 to 10 molar equivalents relative to the amount of the polysaccharide repeating unit 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 polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is 0.1 to 0.5 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

[0283] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

[0284] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is 0.5 to 8 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

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

[0286] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is 0.5 to 4 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

[0287] In one embodiment, step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is 0.5 to 3 molar equivalents relative to the amount of polysaccharide repeating units of the activated polysaccharide.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0305] In one embodiment, step a) further comprises reacting the polysaccharide activated with the carbonic acid derivative with an amount of the azide linker that is 4 to 10 molar equivalents relative to the amount of the polysaccharide repeating unit 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 polysaccharide activated with a carbonic acid derivative with an amount of azide linker that is about 0.5 molar equivalent relative to the amount of polysaccharide repeating units of the activated polysaccharide.

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

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

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

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

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

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

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

[0329] 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 0.5 to 10 molar equivalents relative to the lysine on the carrier.

[0330] 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 10 molar equivalents relative to the lysine on the carrier.

[0331] 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 1.5 to 10 molar equivalents relative to the lysine 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 lysine on the carrier.

[0333] 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 2.5 to 10 molar equivalents relative to the lysine 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 lysine on the carrier.

[0335] 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 5 to 10 molar equivalents relative to the lysine on the carrier.

[0336] 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 7.5 to 10 molar equivalents relative to the lysine on the carrier.

[0337] 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 0.1 to 7.5 molar equivalents relative to the lysine on the carrier.

[0338] 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 0.5 to 7.5 molar equivalents relative to the lysine 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 lysine on the carrier.

[0340] 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 1.5 to 7.5 molar equivalents relative to the lysine 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 lysine on the carrier.

[0342] 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 2.5 to 7.5 molar equivalents relative to the lysine on the carrier.

[0343] In one embodiment, step b) comprises reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3 to 7.5 molar equivalents relative to the lysine 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 lysine 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 lysine 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 lysine 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 lysine 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 lysine 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 lysine on the carrier.

[0350] In one embodiment, step b) involves 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 lysine on the carrier.

[0351] In one embodiment, step b) involves 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 lysine on the carrier.

[0352] In one embodiment, step b) involves 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 lysine on the carrier.

[0353] In one embodiment, step b) involves 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 lysine on the carrier.

[0354] In one embodiment, step b) involves 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 lysine on the carrier.

[0355] In one embodiment, step b) involves 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 lysine on the carrier.

[0356] In one embodiment, step b) involves reacting the carrier protein with an amount of a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2 to 3 molar equivalents relative to the lysine on the carrier.

[0357] In one embodiment, step b) involves 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 lysine on the carrier.

[0358] In one embodiment, step b) involves 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 lysine on the carrier.

[0359] In one embodiment, step b) involves 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 lysine on the carrier.

[0360] In one embodiment, step b) involves 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 lysine on the carrier.

[0361] In one embodiment, step b) involves 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 lysine on the carrier.

[0362] In one embodiment, step b) involves 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 lysine on the carrier.

[0363] In one embodiment, step b) involves 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 lysine 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 a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 2 molar equivalents relative to the lysine on the carrier.

[0379] 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 about 1.5 molar equivalents relative to the lysine on the carrier.

[0380] 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 about 1 molar equivalent relative to the lysine on the carrier.

[0381] 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 about 0.5 molar equivalents relative to the lysine on the carrier.

[0382] 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 about 0.1 molar equivalents relative to the lysine on the carrier.

[0383] In one embodiment, the degree of activation of the activated carrier after step b) is 1 to 50. The degree of activation of the activated carrier is defined as the number of lysine residues in the carrier protein linked to the drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group.

[0384] In one embodiment, the carrier protein is CRM containing 39 lysine residues 197 is. In said embodiment, the degree of activation of the activated carrier after step b) can be 1 to 30. In another embodiment, the activated carrier (CRM 197) The degree of activation is 5 to 20. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is 9 to 18. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is 8 to 11. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is 15 to 20. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 5. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 6. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 7. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 8. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 9. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 10. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 11. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 12. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 13. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 14. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 15. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 16. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 17. In another embodiment, the activated carrier (CRM 197 ) The degree of activation is about 18. In another embodiment, the activated carrier (CRM197 ) has an activation degree of about 19. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 20. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 21. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 22. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 23. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 24. In another embodiment, the activation degree of the activated carrier (CRM 197 ) is about 25.

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

[0386] In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 5 to 50. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 7 to 45. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 5 to 15. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 20 to 30. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 30 to 50. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 30 to 40. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is 10 to 40. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 5. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 7. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 10. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 13. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 15. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 20. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 26. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 30. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 35. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 37. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 40. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 45. In another embodiment, the degree of activation of the activated carrier (SCP) after step b) is about 50.

[0387] In one embodiment, the carrier protein is TT or a fragment thereof. In the said embodiment, the degree of activation of the activated carrier after step b) can be 1 to 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 the activated azide polysaccharide to the activated alkyne carrier in step c) is 0.1 to 3. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is 0.5 to 2. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is 0.6 to 1.5. In a preferred embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is 0.8 to 1. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 0.5. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 0.6. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 0.7. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 0.8. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 0.9. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.1. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.2. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.3. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.4. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.5. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.6.In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.7. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.8. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 1.9. In one embodiment, the initial input ratio (weight to weight) of the activated azide polysaccharide to the activated alkyne carrier in step c) is about 2.

[0391] After the click conjugation reaction, unreacted azide groups may remain in the conjugate, and these can be capped using a suitable azide group capping agent. Thus, in one embodiment, after step c), the unreacted azide groups in the conjugate are capped using a suitable azide group capping agent. In one embodiment, this azide group capping agent is a drug having an alkyne group. In one embodiment, this azide group capping agent is a drug having a terminal alkyne. In one embodiment, this azide group capping agent is a drug having a cycloalkyne.

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

[0393] [wherein X is (CH2) and n is selected from 1 to 15] is a compound. n

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

[0395] Thus, in one embodiment, after step (c), the process further includes the step of capping the unreacted azide groups remaining in the conjugate with an azide group capping agent.

[0396] ​In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.05 to 20 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0397] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.1 to 15 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0398] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.5 to 10 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0399] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.5 to 5 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0400] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.5 to 2 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0401] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.5 to 1 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

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

[0403] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is 0.75 to 1.5 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0404] In one embodiment, the capping of unreacted azide groups is carried out using an amount of a capping agent that is about 1 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

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

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

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

[0408] After the click conjugation reaction, unreacted alkyne groups may remain present in the conjugate, and these can be capped using a suitable alkyne group capping agent. In one embodiment, this alkyne group capping agent is a drug having an azide group.

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

[0410]

Chemical formula

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

[0412] Thus, in one embodiment, after step (c), the process further includes a step of capping unreacted alkyne groups remaining 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 the unreacted alkyne groups is carried out using an amount of a capping agent that is 0.1 to 15 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0415] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 0.5 to 10 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0416] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 0.5 to 5 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0417] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 0.5 to 2 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0418] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 0.5 to 1 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0419] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 1 to 5 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

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

[0421] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is 1.5 to 2.5 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0422] In one embodiment, capping of the unreacted alkyne groups is carried out using an amount of a capping agent that is about 0.5 molar equivalent relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

[0423] In one embodiment, 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, the 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 includes a serotype 3 saccharide conjugatively conjugated with a carrier protein (CP) through a spacer and having the general formula (VII):

[0431] [Chem.] [wherein X is CH2(CH2) n’ , (CH2CH2O) m CH2CH2, NHCO(CH2) n’ , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ , and O(CH2CH2O) m CH2CH2, selected from the group consisting of, n' is selected from 1 to 10, m is selected from 1 to 4, X' is CH2O(CH2) n” CH2C=O, CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, selected from the group consisting of, n'' is selected from 0 to 10, m' is selected from 0 to 4]] and provides a serotype 3 glycoconjugate having the same.

[0432] Formula (VII) is a schematic diagram of the serotype 3 glycoconjugate of the present invention. It should not be understood that the linkage is present in all repeating units of the sugar. Rather, the majority of the Streptococcus pneumoniae serotype 3 sugar repeating units remain unmodified, and the covalent bond between the carrier protein and the sugar is for a minority of the sugar repeating units. Furthermore, a single carrier protein (CP) molecule may be linked to a plurality of Streptococcus pneumoniae serotype 3 sugar molecules, and a single Streptococcus pneumoniae serotype 3 sugar molecule can be linked to more than one carrier protein (CP) molecule.

[0433] In a preferred embodiment, the present invention includes a serotype 3 saccharide conjugatively conjugated with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is CH2(CH2) n’ and n' is 2, X' is CH2O(CH2)n” Provided is a serotype 3 complex carbohydrate having CH2C=O and n” being 1.

[0434] In one embodiment, the present invention includes a serotype 3 sugar conjugated covalently with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is CH2(CH2) n’ and n’ is selected from 1 to 10, and X’ is CH2O(CH2) n”Provided is a serotype 3 complex carbohydrate having CH2C=O, and n” is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5, and n” is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5, and n” is selected from 0 to 5. In one embodiment, n’ is selected from 1 to 3, and n” is selected from 0 to 3. In one embodiment, n’ is selected from 1 to 2, and n” is selected from 0 to 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 a particular embodiment, n' is 1, m' is 0, and n'' is 1. In a particular embodiment, n' is 1, m' is 1, and n'' is 1. In a particular embodiment, n' is 1, m' is 2, and n'' is 1. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 2. In a particular embodiment, n' is 1, m' is 1, and n'' is 2. In a particular embodiment, n' is 1, m' is 2, and n'' is 2. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 3. In a particular embodiment, n' is 1, m' is 1, and n'' is 3. In a particular embodiment, n' is 1, m' is 2, and n'' is 3. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 4. In a particular embodiment, n' is 1, m' is 1, and n'' is 4. In a particular embodiment, n' is 1, m' is 2, and n'' is 4. In a particular embodiment, 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 a particular embodiment, n’ is 1, m’ is 0, and n” is 5. In a particular embodiment, n’ is 1, m’ is 1, and n” is 5. In a particular embodiment, n’ is 1, m’ is 2, and n” is 5. In a particular embodiment, 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 3 sugar conjugated covalently with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is (CH2CH2O) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n”Provided is a serotype 3 complex carbohydrate having the formula CH2C=O, where n” is selected from 0 to 10. 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.

[0468] In one embodiment, the present invention includes a serotype 3 sugar conjugated in common with a carrier protein (CP) through a spacer, having the general formula (VII) [wherein X is (CH2CH2O) m CH2CH2, m is selected from 1 to 4, X’ is 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 complex carbohydrate having the same.

[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 a specific embodiment, m is 1, m’ is 0, and n” is 0. In another embodiment, m is 1, m’ is 1, and n” is 0. In another embodiment, m is 1, m’ is 2, and n” is 0. In another embodiment, 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 a specific embodiment, m is 1, m' is 0, and n" is 1. In a specific embodiment, m is 1, m' is 1, and n" is 1. In a specific embodiment, m is 1, m' is 2, and n" is 1. In a specific embodiment, 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 further embodiments, m is 4, m' is 0, and n" is 2. In further embodiments, m is 4, m' is 1, and n" is 2. In further embodiments, m is 4, m' is 2, and n" is 2. In further embodiments, 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 a particular embodiment, m is 1, m' is 0, and n'' is 4. In a particular embodiment, m is 1, m' is 1, and n'' is 4. In a particular embodiment, m is 1, m' is 2, and n'' is 4. In a particular embodiment, 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 invention comprises a serotype 3 sugar conjugated jointly with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is NHCO(CH2) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2)n”Provided is a serotype 3 complex carbohydrate having CH2C=O, where n’ is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5 and n” is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5 and n” is selected from 0 to 5. In one embodiment, n’ is selected from 1 to 3 and n” is selected from 0 to 3. In one embodiment, n’ is selected from 1 to 2 and n” is selected from 0 to 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 invention provides a serotype 3 complex carbohydrate comprising a serotype 3 sugar conjugated covalently with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is NHCO(CH2) n’ and n' is selected from 1 to 10, X' is CH2O(CH2CH2O) 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 another embodiment, n' is 1, m' is 1, and n'' is 0. In another embodiment, n' is 1, m' is 2, and n'' is 0. In another embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n" is 1. In a particular embodiment, n' is 1, m' is 1, and n" is 1. In a particular embodiment, n' is 1, m' is 2, and n" is 1. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 2. In a particular embodiment, n' is 1, m' is 1, and n'' is 2. In a particular embodiment, n' is 1, m' is 2, and n'' is 2. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 3. In a particular embodiment, n' is 1, m' is 1, and n'' is 3. In a particular embodiment, n' is 1, m' is 2, and n'' is 3. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n" is 4. In a particular embodiment, n' is 1, m' is 1, and n" is 4. In a particular embodiment, n' is 1, m' is 2, and n" is 4. In a particular embodiment, 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 3 sugar conjugated covalently with a carrier protein (CP) through a spacer, and has the general formula (VII) [wherein X is NHCO(CH2CH2O) m CH2CH2, m is selected from 1 to 4, and X' is CH2O(CH2) n”Provided is a serotype 3 complex carbohydrate having the formula CH2C=O, where n” is selected from 0 to 10. 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.

[0528] In one embodiment, the present invention includes a serotype 3 sugar conjugated in common with a carrier protein (CP) through a spacer, having the general formula (VII) [wherein X is NHCO(CH2CH2O) m CH2CH2, m is selected from 1 to 4, X' is 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 complex carbohydrate having the formula.

[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 a specific embodiment, m is 1, m' is 0, and n'' is 0. In another embodiment, m is 1, m' is 1, and n'' is 0. In another embodiment, m is 1, m' is 2, and n'' is 0. In another embodiment, 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 further embodiments, m is 4, m' is 0, and n'' is 0. In further embodiments, m is 4, m' is 1, and n'' is 0. In further embodiments, m is 4, m' is 2, and n'' is 0. In further embodiments, 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 further embodiments, m is 4, m' is 0, and n'' is 1. In further embodiments, m is 4, m' is 1, and n'' is 1. In further embodiments, m is 4, m' is 2, and n'' is 1. In further embodiments, 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 further embodiments, m is 4, m' is 0, and n" is 2. In further embodiments, m is 4, m' is 1, and n" is 2. In further embodiments, m is 4, m' is 2, and n" is 2. In further embodiments, 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 a particular embodiment, m is 1, m' is 0, and n'' is 4. In a particular embodiment, m is 1, m' is 1, and n'' is 4. In a particular embodiment, m is 1, m' is 2, and n'' is 4. In a particular embodiment, 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 invention comprises a serotype 3 sugar conjugated to a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is OCH2(CH2) n’ and n' is selected from 1 to 10, and X' is CH2O(CH2)n”Provided is a serotype 3 complex carbohydrate having CH2C=O, and n” is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5 and n” is selected from 0 to 10. In one embodiment, n’ is selected from 1 to 5 and n” is selected from 0 to 5. In one embodiment, n’ is selected from 1 to 3 and n” is selected from 0 to 3. In one embodiment, n’ is selected from 1 to 2 and n” is selected from 0 to 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 provides a serotype 3 complex carbohydrate comprising a serotype 3 sugar conjugated covalently with a carrier protein (CP) through a spacer and having the general formula (VII) [wherein X is OCH2(CH2) n’ and n' is selected from 1 to 10, X' is CH2O(CH2CH2O) m’ (CH2) n” CH2C=O, and 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 another embodiment, n' is 1, m' is 1, and n'' is 0. In another embodiment, n' is 1, m' is 2, and n'' is 0. In another embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 1. In a particular embodiment, n' is 1, m' is 1, and n'' is 1. In a particular embodiment, n' is 1, m' is 2, and n'' is 1. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 2. In a particular embodiment, n' is 1, m' is 1, and n'' is 2. In a particular embodiment, n' is 1, m' is 2, and n'' is 2. In a particular embodiment, 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 a particular embodiment, n' is 1, m' is 0, and n'' is 4. In a particular embodiment, n' is 1, m' is 1, and n'' is 4. In a particular embodiment, n' is 1, m' is 2, and n'' is 4. In a particular embodiment, 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 a specific embodiment, n' is 1, m' is 0, and n" is 5. In a specific embodiment, n' is 1, m' is 1, and n" is 5. In a specific embodiment, n' is 1, m' is 2, and n" is 5. In a specific embodiment, 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 3 saccharide conjugated covalently through a spacer to a carrier protein (CP) and having the general formula (VII) [wherein X is O(CH2CH2O) mIt is CH2CH2, m is selected from 1 to 4, and X’ is CH2O(CH2CH2O) m’ (CH2) n” It is CH2C=O, n” is selected from 0 to 10, and m’ is selected from 0 to 4], and provides a serotype 3 complex carbohydrate having the same.

[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 a specific embodiment, m is 1, m’ is 0, and n” is 0. In another embodiment, m is 1, m’ is 1, and n” is 0. In another embodiment, m is 1, m’ is 2, and n” is 0. In another embodiment, 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 a particular embodiment, m is 1, m' is 0, and n" is 1. In a particular embodiment, m is 1, m' is 1, and n" is 1. In a particular embodiment, m is 1, m' is 2, and n" is 1. In a particular embodiment, 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 a particular embodiment, m is 1, m' is 0, and n" is 2. In a particular embodiment, m is 1, m' is 1, and n" is 2. In a particular embodiment, m is 1, m' is 2, and n" is 2. In a particular embodiment, 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 a particular embodiment, m is 1, m' is 0, and n'' is 3. In a particular embodiment, m is 1, m' is 1, and n'' is 3. In a particular embodiment, m is 1, m' is 2, and n'' is 3. In a particular embodiment, 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 a particular embodiment, m is 1, m' is 0, and n" is 4. In a particular embodiment, m is 1, m' is 1, and n" is 4. In a particular embodiment, m is 1, m' is 2, and n" is 4. In a particular embodiment, 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 a specific embodiment, m is 1, m' is 0, and n" is 5. In a specific embodiment, m is 1, m' is 1, and n" is 5. In a specific embodiment, m is 1, m' is 2, and n" is 5. In a specific embodiment, 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 protein of the Streptococcus pneumoniae serotype 3 complex carbohydrate of the present 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 107; and other mutations described by Nicholls and Youle, Genetically Engineered Toxins, Frankel ed., Maecel Dekker Inc., (1992); deletion of Glu-148 or mutation to Asp, Gln, or Ser and / or deletion of Ala158 or mutation to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutation of at least one or more of the residues Lys 516, Lys 526, Phe 530, and / or Lys 534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017 and 6,455,673; or a fragment disclosed in U.S. Pat. No. 5,843,711), ply detoxified in some manner, e.g., dPLY-GMBS (WO2004 / 081515, WO2006 / 032499) or pneumococcal pneumolysin (ply) including dPLY-formol (Kuo et al. (1995) Infect lmmun, 63:2706-2713), PhtX including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD, or PhtE are disclosed in WO00 / 37105 and WO00 / 39299), and protein fusions, e.g., PhtDE fusion, PhtBE fusion, Pht A-E (WO01 / 98334, WO03 / 054007, WO2009 / 000826), usually outer membrane protein complex (OMPC) (Neisseria meningitidis outer membrane protein) extracted from Neisseria meningitidis serogroup B (EP0372501), PorB (Neisseriaderived from meningitidis), PD (Haemophilus influenzae protein D, see, for example, EP0594610 B), or an immunologically functional equivalent thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO93 / 17712, WO94 / 03208), pertussis proteins (WO98 / 58668, EP0471177), cytokines, lymphokines, growth factors, or hormones (WO91 / 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), such as the N19 protein (Baraldoi et al. (2004) Infect Immun, 72:4884-4887), pneumococcal surface protein PspA (WO02 / 091998), iron uptake proteins (WO01 / 72337), toxin A or B of Clostridium difficile (WO00 / 61761), transferrin-binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially its non-toxic mutants such as exotoxin A having a substitution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol., 169(11):4967-4971)) are selected from the group consisting of. 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 (described, for example, in WO2004 / 083251), Escherichia coli LT, Escherichia coli ST, and exotoxin A from Pseudomonas aeruginosa. Another suitable carrier protein is C5a peptidase (SCP) from the genus 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 The protein is a safe and effective T cell-dependent carrier for sugars. CRM 197 And further details regarding its production can be found, for example, in U.S. Patent No. 5,614,382.

[0622] In one embodiment, the serotype 3 capsular polysaccharide is conjugated to the CRM 197 protein. In one embodiment, the serotype 3 capsular polysaccharide is conjugated to the CRM 197 protein or the A chain of CRM 197 (see CN103495161). In one embodiment, the serotype 3 capsular polysaccharide is conjugated to the A chain of CRM obtained through expression by genetically recombinant Escherichia coli (E. coli) (see CN103495161). 197 (see CN103495161).

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

[0624] Two important species of β-hemolytic streptococci, namely Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS), which cause a variety of severe human infections ranging from mild pharyngitis and impetigo to severe invasive diseases such as necrotizing fasciitis (GAS) and neonatal sepsis (GBS), have evolved ways to break 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 a presequence of the export signal and are removed upon passage through the cytoplasmic membrane. The next 68 residues serve as a prosequence and must be removed to generate active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting from Lys-1034, there are four consecutive 17-residue motifs, followed by a cell sorting and cell wall attachment signal. 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] SCP can be divided into domains (see Figure 1B in Brown et al., PNAS, 2005, Vol. 102, No. 51, pages 18391 - 18396). These domains are the pre / prodomain (including the presequence of the export signal (generally the first 31 residues) and the prosequence (generally the next 68 residues)), the protease domain (split into two parts (protease part 1 is generally residues 89 - 333 / 334, and protease domain part 2 is generally residues 467 / 468 - 583 / 584)), the protease - associated domain (PA domain) (generally residues 333 / 334 - 467 / 468), three fibronectin type III (Fn) domains (Fn1, generally residues 583 / 584 - 712 / 713, Fn2, generally residues 712 / 713 - 928 / 929 / 930, generally Fn3, residues 929 / 930 - 1029 / 1030 / 1031), and the cell - wall anchor domain (generally the 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 (SCPB) from GBS. Examples of SCPB are provided by SEQ ID NO: 3 in WO97 / 26008. See also SEQ ID NO: 3 in WO00 / 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 SCPA can be found in SEQ ID NO: 1 and SEQ ID NO: 2 in WO97 / 26008. See also SEQ ID NO: 1, 2, and 23 in WO00 / 34487.

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

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

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

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

[0633] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is a fragment of SCP that includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal.

[0634] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is a fragment of SCP that includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal.

[0635] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP that includes two of a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal.

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

[0643] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, the replacement of the at least one amino acid is in the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement is D130A. In another embodiment, the replacement is H193A. In another embodiment, the replacement is N295A. In yet another embodiment, the replacement is S512A.

[0644] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is an enzymatically inactive fragment of SCP that contains a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not contain a presequence, a prosequence, and a cell wall anchor domain of an export signal, and the inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, the replacement of the at least one amino acid is in the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement is D130A. In another embodiment, the replacement is H193A. In another embodiment, the replacement is N295A. In yet another embodiment, the replacement 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 that contains a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not contain a presequence, a prosequence, and a cell wall anchor domain of an export signal, and the inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, the replacement of the at least one amino acid is in the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement is D130A. In another embodiment, the replacement is H193A. In another embodiment, the replacement is N295A. In yet another embodiment, the replacement is S512A.

[0646] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention comprises a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include a presequence of an export signal, a prosequence, and a cell wall anchor domain, and is an enzymatically inactive fragment of SCPB, and the inactivation is achieved by replacing at least one amino acid of the wild-type sequence. Preferably, the replacement of the at least one amino acid is in the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement is D130A. In another embodiment, the replacement is H193A. In another embodiment, the replacement is N295A. In yet another embodiment, the replacement is S512A.

[0647] In one embodiment, the enzymatic activity of SCP is inactivated by replacing at least two amino acids of the wild-type sequence. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. In one embodiment, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0648] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is an enzymatically inactive SCP, and the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0649] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is enzymatically inactive SCPA, and the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0650] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide complex carbohydrate of the present invention is enzymatically inactive SCPB, and the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0651] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0652] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention comprises a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal, and is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0653] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention comprises a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal, and is an enzymatically inactive fragment of SCPA, wherein the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0654] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention comprises a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the pre-sequence, pro-sequence, and cell wall anchor domain of the export signal, and is an enzymatically inactive fragment of SCPB, wherein the inactivation is achieved by replacing at least two amino acids of the wild-type sequence. Preferably, the replacement of the at least two amino acids is in the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least two amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least two amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least two amino acids is D130A and H193A. In one embodiment, the replacement of the at least two amino acids is D130A and N295A. Preferably, the replacement of the at least two amino acids is D130A and S512A. In one embodiment, the replacement of the at least two amino acids is H193A and N295A. In one embodiment, the replacement of the at least two amino acids is H193A and S512A. In one embodiment, the replacement of the at least two amino acids is N295A and S512A.

[0655] In one embodiment, the enzymatic activity of SCP is inactivated by replacing at least three amino acids of the wild-type sequence. In one embodiment, the replacement of said at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of said at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of said at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of said at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of said at least three amino acids is H193A, N295A, and S512A.

[0656] Thus, in one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive SCP, and said inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of said at least three amino acids is in the protease domain. In one embodiment, the replacement of said at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of said at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of said at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of said at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of said at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of said at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of said at least three amino acids is H193A, N295A, and S512A.

[0657] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is enzymatically inactive SCPA, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of the at least three amino acids is in the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is H193A, N295A, and S512A.

[0658] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is enzymatically inactive SCPB, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of the at least three amino acids is in the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is H193A, N295A, and S512A.

[0659] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of the at least three amino acids is in the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is H193A, N295A, and S512A.

[0660] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include a presequence of an export signal, a prosequence, and a cell wall anchor domain, and is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the substitution of the at least three amino acids is in the protease domain. In one embodiment, the substitution of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the substitution of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the substitution of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the substitution of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the substitution of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the substitution of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the substitution of the at least three amino acids is H193A, N295A, and S512A.

[0661] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention comprises a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include a presequence of an export signal, a prosequence, and a cell wall anchor domain, and is an enzymatically inactive fragment of SCPA, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of the at least three amino acids is in the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is H193A, N295A, and S512A.

[0662] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide complex carbohydrate of the present invention includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include a presequence of an export signal, a prosequence, and a cell wall anchor domain, and is an enzymatically inactive fragment of SCPB, and the inactivation is achieved by replacing at least three amino acids of the wild-type sequence. Preferably, the replacement of the at least three amino acids is in the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least three amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least three amino acids is selected from the group consisting of D130A, H193A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and N295A. In one embodiment, the replacement of the at least three amino acids is D130A, H193A, and S512A. In one embodiment, the replacement of the at least three amino acids is D130A, N295A, and S512A. In one embodiment, the replacement of the at least three amino acids is H193A, N295A, and S512A.

[0663] In one embodiment, the enzymatic activity of SCP is inactivated by replacing at least four amino acids of the wild-type sequence. In one embodiment, the replacement of the 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 conjugate of the present invention is an enzymatically inactive SCP, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0665] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive SCPA, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0666] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive SCPB, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A. [[ID=A]] [[ID=B]]

[0667] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0668] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP that includes a protease domain, a protease-associated domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0669] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCPA that includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least one amino acid is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0670] In one embodiment, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCPB that includes a protease domain, a protease-related domain (PA domain), and three fibronectin type III (Fn) domains, but does not include the presequence, prosequence, and cell wall anchor domain of the export signal, and the inactivation is achieved by replacing at least four amino acids of the wild-type sequence. Preferably, the replacement of the at least four amino acids is in the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 1 of the protease domain. In one embodiment, the replacement of the at least four amino acids is in part 2 of the protease domain. In one embodiment, the replacement of the at least four amino acids is D130A, H193A, N295A, and S512A.

[0671] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of SEQ ID NO: 41.

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

[0673] SEQ ID NO: 41: MAKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTDKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

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

[0675] SEQ ID NO: 42: AKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNHEAWRLTDKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKTAVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLADYARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGVSIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQLTETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGKIALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAFISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKPDIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMTPSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYVTDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKHFALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLEGFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEANSDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESSEITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGTFLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTRWDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATSATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTFTLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ

[0676] Accession number 42 has a length of 949 amino acids.

[0677] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity with SEQ ID NO: 41.

[0678] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity with SEQ ID NO: 41.

[0679] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99% identity with SEQ ID NO: 41.

[0680] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity with SEQ ID NO: 41.

[0681] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity with SEQ ID NO: 41.

[0682] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity with SEQ ID NO: 41.

[0683] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide conjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity with SEQ ID NO: 42.

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

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

[0686] In certain embodiments, the carrier protein of the serotype 3 capsular polysaccharide glycoconjugate of the present invention is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity with SEQ ID NO: 42.

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

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

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

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

[0691] In one embodiment, the present invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 3 complex carbohydrate of the present invention and 1 to 25 complex carbohydrates (1 to 25 pneumococcal conjugates) from various serotypes of Streptococcus pneumoniae (S. pneumoniae). In one embodiment, the present invention relates to an immunogenic composition comprising complex carbohydrates from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different serotypes of Streptococcus pneumoniae (S. pneumoniae). In one embodiment, the immunogenic composition comprises complex carbohydrates from 16 or 20 different serotypes of Streptococcus pneumoniae (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 the Streptococcus pneumoniae serotype 3 polysaccharide and further comprising polysaccharides from Streptococcus pneumoniae (S. pneumoniae) serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

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

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

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

[0697] In one embodiment, any of the above immunogenic compositions further comprises polysaccharides from St...

Claims

**Claim 1** (a) A step of reacting an isolated Streptococcus pneumoniae serotype 3 capsular polysaccharide with a carbonate derivative and an azide linker in an aprotic solvent to produce an activated azide polysaccharide, wherein the carbonate derivative is 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), or N-hydroxysuccinimidyl chloroformate; (b) A step of reacting a carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, wherein the NHS moiety reacts with an amino group to form an amide bond, thereby obtaining an alkyne-functionalized carrier protein; (c) Cu ＋１ A step of reacting the activated azide polysaccharide of step (a) with the activated alkyne carrier protein of step (b) by a mediated azide-alkyne cycloaddition reaction to form a glycoconjugate A method for preparing a Streptococcus pneumoniae serotype 3 glycoconjugate, comprising the above steps. **Claim 2** The method according to claim 1, wherein the isolated polysaccharide is sized before the activation step (a). **Claim 3** The method according to claim 2, wherein the isolated serotype 3 capsular polysaccharide is sized to a weight average molecular weight of 100 kDa to 200 kDa. **Claim 4** The azide linker is of formula (I): 【Chemical 1】 [wherein, X is CH ２ (CH ２ ) ｎ , (CH ２ CH ２ O) ｍ CH ２ CH ２ , NHCO(CH ２ ) ｎ , NHCO(CH ２ CH ２ O) ｍ CH ２ CH ２ , OCH ２ (CH ２ ) ｎ , and O(CH ２ CH ２ O) ｍ CH ２ CH ２ ; n is selected from 1 to 10; and m is selected from 1 to 4], the method according to any one of claims 1 to 3. **Claim 5** The azide linker is of formula (II): [Chemical 2] The method according to any one of claims 1 to 3, wherein the azide linker is a compound of the above formula. **Claim 6** The method according to any one of claims 1 to 5, wherein the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne. **Claim 7** The method according to any one of claims 1 to 5, wherein the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent having an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne. **Claim 8** The method according to any one of claims 1 to 5, wherein the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is of formula (III): [Chemical 3] [wherein, X is CH ２ O(CH ２ ) ｎ CH ２ C=O and CH ２ O(CH ２ CH ２ O) ｍ (CH ２ ) ｎ CH ２ C=O, n is selected from 0 to 10, and m is selected from 0 to 4] The method according to any one of claims 1 to 5, wherein the agent is a compound of the above formula. **Claim 9** The method according to any one of claims 1 to 8, wherein the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is of formula (IV): 【Chemical 4】 The method according to any one of claims 1 to 8, wherein the agent is a compound of the above formula. **Claim 10** The method according to any one of claims 1 to 9, wherein step a) further comprises reacting the polysaccharide activated with a carbonic acid derivative with an amount of an azide linker that is 0.01 to 10 molar equivalents relative to the amount of the polysaccharide repeating unit of the activated polysaccharide.

11. The method according to any one of claims 1 to 10, wherein 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 0.1 to 10 molar equivalents relative to the lysine on the carrier.

12. The method according to any one of claims 1 to 11, wherein the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst.

13. The method according to any one of claims 1 to 12, further comprising, after step c), capping the unreacted azide groups remaining in the conjugate with an azide group capping agent.

14. The method according to any one of claims 1 to 13, further comprising, after step c), capping the unreacted alkyne groups remaining in the conjugate with an alkyne group capping agent.

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

16. Containing Streptococcus pneumoniae serotype 3 polysaccharide covalently conjugated with a carrier protein (CP) through a spacer, of the general formula (VII): 【Chemical 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 ; n' is selected from 1 to 10; 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 selected from the group consisting of C=O, n" is selected from 0 to 10, and m' is selected from 0 to 4] A Streptococcus pneumoniae serotype 3 glycoconjugate having

17. The serotype 3 glycoconjugate according to claim 16, wherein the weight average molecular weight (Mw) of the polysaccharide before conjugation is 10 kDa to 2000 kDa.

18. Containing Streptococcus pneumoniae serotype 3 sugar conjugated in common with a carrier protein (CP) through a spacer, having the general formula (VII) [wherein X is CH ２ (CH ２ ) ｎ’ and n' is 2, X' is CH ２ O(CH ２ ) ｎ” CH ２ C=O and n'' is 1], a Streptococcus pneumoniae serotype 3 complex carbohydrate.

19. The serotype 3 glycoconjugate according to any one of claims 16 to 18, containing serotype 3 capsular polysaccharide, wherein the weight average molecular weight (Mw) of the polysaccharide before conjugation is 75 kDa to 200 kDa.

20. The serotype 3 glycoconjugate according to any one of claims 16 to 19, having a weight average molecular weight (Mw) of 1000 kDa to 4000 kDa.

21. The carrier protein is CRM １９７ The serotype 3 complex carbohydrate according to any one of claims 16 to 20

22. The serotype 3 glycoconjugate according to any one of claims 16 to 20, wherein the carrier protein is SCP.

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