Immunogenic composition containing conjugate-encapsulated sugar antigen and its use

Abstract

The present invention relates to a novel immunogenic composition containing a conjugated Streptococcus pneumoniae capsular sugar antigen (sugar conjugate), a kit containing the immunogenic composition, and the use of the same. The immunogenic composition of the present invention typically contains at least one sugar conjugate derived from a Streptococcus pneumoniae serotype not found in PREVNAR®, SYNFLORIX®, and / or PREVNAR13®. The present invention also relates to the vaccination of human subjects, particularly infants and the elderly, against pneumococcal infection using the novel immunogenic composition.

Description

[Technical Field]

[0001] This invention relates to immunogenic compositions and vaccines, their manufacture, and the use of such compositions in medicine.

[0002] More specifically, the present invention relates to isolated Streptococcus pneumoniae serotype 21 sugars, their sugar conjugates, methods for preparing Streptococcus pneumoniae serotype 21 sugar conjugates, and immunogenic compositions containing Streptococcus pneumoniae serotype 21 sugar conjugates.

[0003] The present invention also relates to analytical methods for analyzing isolated Streptococcus pneumoniae serotype 21 polysaccharides, reduced serotype 21 polysaccharides, or Streptococcus pneumoniae serotype 21 sugar conjugates.

[0004] The Streptococcus pneumoniae serotype 21 sugar and sugar conjugate of the present invention can be used as a vaccine. [Background technology]

[0005] For decades, techniques have been successfully used to increase the immunogenicity of low-immunogenic molecules by conjugating them to "carrier" molecules (e.g., Goebel et al. (1939) J.Exp.Med.69:53). For example, many immunogenic compositions have been described that utilize this "carrier effect" by conjugating purified capsule 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 bypass the weak antibody response usually observed in infants when immunized with free polysaccharides (Anderson et al. (1985) J.Pediatr.107:346; Insel et al. (1986) J.Exp.Med.158:294).

[0006] Conjugates have been successfully generated using various crosslinking or coupling reagents, such as homobifunctional, heterobifunctional, or zero-length crosslinking agents. Many methods are currently available for coupling immunogenic molecules, such as sugars, proteins, and peptides, to peptide or protein carriers. Many methods create amine, amide, urethane, isothiourea, or disulfide bonds, or in some cases, thioethers. A drawback of using crosslinking or coupling reagents that introduce reaction sites into the side chains of reactive amino acid molecules on the carrier and / or immunogenic molecule is that, if not neutralized, the reaction sites are free to react with any undesirable molecules in vitro or in vivo (in vitro, thus potentially adversely affecting the function or stability of the conjugate; in vivo, thus posing a potential risk of adverse events in humans or animals immunized with the preparation). Various known chemical reactions can be used to react or "capp" such excess reaction sites to inactivate them; otherwise, these reactions can be destructive to the function of the conjugate.

[0007] Thus, there is still a need for novel, appropriately capped glycoconjugates and methods for preparing such conjugates, such that their function is preserved and the conjugate retains its ability to elicit a desired immune response.

[0008] Pneumococcal polysaccharides, particularly capsular polysaccharides, are important immunogens found on the surface of bacteria. For this reason, they are important components in the design of pneumococcal vaccines. They have been shown to be particularly useful in eliciting an immune response when linked to carrier proteins. [Overview of the project] [Problems that the invention aims to solve]

[0009] Thus, an antigen capable of generating a robust immune response against Streptococcus pneumoniae serotype 21 is needed.

[0010] The present invention provides, in particular, a Streptococcus pneumoniae serotype 21 glycoconjugate. [Means for solving the problem]

[0011] To satisfy these and other needs, the present invention provides the following iterative units:

[0012] [ka] (In the formula, n represents the number of iteration units.) This relates to isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugars.

[0013] In one embodiment, the present invention comprises the following iterative units:

[0014] [ka] (In the formula, n represents the number of repeating units; the O-acetyl group at position 3 of β-D-Galf is present in approximately 50% to 80% of the repeating units; the O-acetyl group at position 6 of β-D-Galf is present in approximately 20% to 40% of the repeating units; and the O-acetyl group at position 5 of β-D-Galf is present in approximately 1% to 10% of the repeating units.) This relates to isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugars.

[0015] In certain embodiments, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar of the present invention has 10 to 5,000 repeat units.

[0016] In one embodiment, the present invention provides a sugar conjugate comprising a sugar having the repeating units disclosed above, conjugated to a carrier protein.

[0017] In one embodiment, the present invention relates to a method for preparing such a conjugate.

[0018] In one embodiment, the present invention relates to an immunogenic composition comprising the Streptococcus pneumoniae serotype 21 sugar and / or the Streptococcus pneumoniae serotype 21 sugar conjugate of the present invention.

[0019] The present invention further relates to a method for analyzing serotype 21 sugars and conjugates of Streptococcus pneumoniae (S. pneumoniae). [Brief explanation of the drawing]

[0020] [Figure 1] This is a schematic diagram of the repeating unit structure of Streptococcus pneumoniae polysaccharide serotype 21. [Figure 2] This figure shows the 1D 1H spectra of serotype 21 polysaccharides (the inset spectrum shows the enlarged anomeric region). The inset table shows the normalized and expected peak areas for the anomeric and methyl protons. The anomeric and methyl signals are annotated. [Figure 3]This figure shows the expanded anomeric regions of the proton-decoupled 1H-13C HSQC spectra of serotype 21 polysaccharides. The coupling constants 1JCH for each sugar are annotated. [Figure 4] This figure shows the quantification of O-acetylation transfer. The bottom, middle, and top panels show enlarged regions of the 1D 1H spectra of serotype 21 at 0, 9, and 15 hours, respectively. All peaks are annotated. The top scheme shows the transfer of O-acetyl from position 3 to 6 of the β-D-Galf sugar. [Figure 5] This figure shows the quantification of O-acetylation transfer. The bottom, middle, and top panels show enlarged regions of the 1D 1H spectra of serotype 21 at days 0, 9, and 14, respectively. All peaks are annotated. The top scheme shows the transfer of O-acetyl from position 3 to 6 of the β-D-Galf sugar. [Figure 6] This figure shows the 1D 1H spectra of the enlarged anomeric and methyl regions of O-acetylated (lower panel) and de-O-acetylated (upper panel) serotype 21 polysaccharides. The anomeric and methyl signals are annotated for both spectra. Shaded boxes indicate resonances observed due to O-acetylation of the β-D-Galf residue, which are absent in the de-O-acetylated serotype 21 polysaccharide. [Figure 7] This is a chemical shift difference plot of O-acetylated and de-O-acetylated serotype 21 polysaccharides (left panel). The effect of deacetylation on the structure of serotype 21 polysaccharides is shown by the black spheres on the structure of the serotype 21 polysaccharide. Arrows indicate bars representing deacetylation resonance. [Modes for carrying out the invention]

[0021] This invention is in part based on the identification of a novel pneumococcal polysaccharide structure using NMR spectroscopy. The structure provided herein is considered to be the first or first accurate identification of Streptococcus pneumoniae serotype 21.

[0022] Using the produced (and purified) polysaccharides, polysaccharide-protein conjugates (glycoconjugates) were generated. Serotype 21 of Streptococcus pneumoniae has a unique polysaccharide structure, which provides unique consideration when designing the conjugate production process. The Streptococcus pneumoniae serotype 21 glycoconjugate of the present invention also has a unique structure and design.

[0023] 1. The isolated Streptococcus pneumoniae serotype 21 sugar of the present invention. As used herein, the term “isolated” in relation to sugars refers to the isolation of Streptococcus pneumoniae (S. pneumoniae) serotype-specific capsular polysaccharides from purified polysaccharides using purification techniques known in the art, including centrifugation, deep filtration, sedimentation, ultrafiltration, treatment with activated carbon, diafiltration, and / or column chromatography. Generally, isolated polysaccharides refer to the partial removal of proteins, nucleic acids, and nonspecific endogenous polysaccharides (C-polysaccharides). Isolated polysaccharides contain less than 10%, 8%, 6%, 4%, or 2% protein impurities and / or nucleic acids. Isolated polysaccharides contain less than 20% C-polysaccharides with respect to type-specific polysaccharides.

[0024] Throughout this specification, the term “sugar” may refer to polysaccharides or oligosaccharides, and may include both. In preferred embodiments, the sugar is a polysaccharide, in particular the capsular polysaccharide of Streptococcus pneumoniae serotype 21.

[0025] Throughout this specification, the terms "serotype 21 sugar," "serotype 21 capsular sugar," and "Streptococcus pneumoniae serotype 21 sugar" refer to Streptococcus pneumoniae serotype 21 capsular sugar and can be used interchangeably.

[0026] The structure of the serotype 21 capsular polysaccharide of Streptococcus pneumoniae (S. pneumoniae) has been disclosed for the first time and is shown in Figure 1. The inventors have found that the serotype 21 polysaccharide consists of hexasaccharides: α-D-galactopyranose (A), β-L-rhamnose (B), β-D-glucose (C), β-D-glucosamine (D), α-D-galactopyranose (E), and O-acetylated β-D-galactofuranose (F).

[0027] The inventors also found that the O-acetylation of β-D-galactofuranose (residue F) can occur at three different positions: positions 3, 5, and 6. Thus, three types of repeat units constitute the natural serotype 21 polysaccharide: a repeat unit O-acetylated at position C3 of the β-D-galactofuranose residue (the most abundant type of repeat unit in the polysaccharide), a repeat unit O-acetylated at position C6 of the β-D-galactofuranose residue (the repeat unit of intermediate abundance in the polysaccharide), and a repeat unit O-acetylated at position C5 of the β-D-galactofuranose residue (the repeat unit of low abundance in the polysaccharide).

[0028] Therefore, in one embodiment, the present invention has the following iteration units:

[0029] [ka] (In the formula, n represents the number of iteration units.) This provides isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugars.

[0030] As shown in Figure 1, the β-D-galactofuranose (residue F) of serotype 21 polysaccharide is O-acetylated at positions 3, 5, and 6 of carbon atoms.

[0031] Therefore, serotype 21 polysaccharide is composed of three types of repeating units: a repeating unit O-acetylated at the C3 position of the β-D-Galf residue, a repeating unit O-acetylated at the C5 position of the β-D-Galf residue, and a repeating unit O-acetylated at the C6 position of the β-D-Galf residue.

[0032] Therefore, in one embodiment, the present invention has the following iteration units:

[0033] [ka] (In the formula, n represents the number of repeating units; the O-acetyl group at position 3 of β-D-Galf is present in approximately 50% to 80% of the repeating units; the O-acetyl group at position 6 of β-D-Galf is present in approximately 20% to 40% of the repeating units; and the O-acetyl group at position 5 of β-D-Galf is present in approximately 1% to 10% of the repeating units.) This provides isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugars having the following characteristics: Preferably, all repeating units have one O-acetyl group on a β-D-Galf residue.

[0034] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 60% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 30% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 10% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0035] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 64% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 30% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 6% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0036] Furthermore, migration of the O-acetyl group from position 3 to position 6 of the β-D-Galf sugar was observed (see Example 3). Accordingly, in one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 25% to about 60% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 30% to about 65% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 5% to about 15% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0037] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 50% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 40% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 10% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0038] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 47% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 42% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 11% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0039] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 30% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 60% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 10% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0040] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, the O-acetyl group at position 3 of β-D-Galf is present in about 31% of the repeat units, the O-acetyl group at position 6 of β-D-Galf is present in about 61% of the repeat units, and the O-acetyl group at position 5 of β-D-Galf is present in about 8% of the repeat units). Preferably, all repeat units have one O-acetyl group on the β-D-Galf residue.

[0041] Furthermore, the naturally occurring serotype 21 polysaccharide of Streptococcus pneumoniae can be deacetylated, for example, by treatment with a base (alkaline pH). As shown in the Examples section, the naturally occurring serotype 21 polysaccharide can be completely deacetylated after incubation with 0.25N NH4OH at room temperature for 24 hours.

[0042] Therefore, in one embodiment, the present invention has the following iteration units:

[0043] [ka] (In the formula, n represents the number of iteration units.) This provides isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugars.

[0044] Naturally occurring serotype 21 sugars of Streptococcus pneumoniae can be partially deacetylated, for example, by mild treatment with a base (alkaline pH).

[0045] Accordingly, in one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, and the O-acetyl groups at the 3, 5, or 6 positions of β-D-Galf are present in about 0% to about 95% of the repeat units).

[0046] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, and the O-acetyl groups at the 3, 5, or 6 positions of β-D-Galf are present in about 10% to about 90% of the repeat units).

[0047] In one embodiment, the present invention provides an isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, and the O-acetyl groups at the 3, 5, or 6 positions of β-D-Galf are present in about 30% to about 60% of the repeat units).

[0048] In certain embodiments, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar of the present invention has 10 to 5,000 repeat units. In certain embodiments, the isolated sugar has 50 to 4,500 repeat units. In certain embodiments, the isolated sugar has 100 to 4,500 repeat units. In certain embodiments, the isolated sugar has 150 to 2,000 repeat units.

[0049] Isolated capsular sugars derived from serotype 21 of Streptococcus pneumoniae can be prepared by standard techniques known to those skilled in the art. Typically, capsular polysaccharides are produced by growing serotype 21 strains of Streptococcus pneumoniae in a culture medium (e.g., a soybean-based medium), and then the polysaccharides are prepared from the bacterial culture. Serotype 21 strains of Streptococcus pneumoniae can be obtained from established strain storage facilities (e.g., Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA)) or clinical specimens.

[0050] Populations of the organism (Streptococcus pneumoniae serotype 21) are often scaled up from seed vials to seed bottles and subcultured through one or more seed fermenters, increasing the volume until production-scale fermentation capacity is reached. At the end of the growth cycle, the cells are lysed, and the lysate broth is collected for downstream (purification) processing (see, for example, WO2006 / 110381 and WO2008 / 118752, U.S. Patent Applications Publications 2006 / 0228380, 2006 / 0228381, 2008 / 0102498 and 2008 / 0286838). Polysaccharides are typically purified by centrifugation, precipitation, ultrafiltration, and / or column chromatography (see, e.g., WO2006 / 110352, WO2008 / 118752, and WO2020 / 170190).

[0051] Isolated polysaccharides can be characterized by various parameters, such as molecular average molecular weight (Mw). The molecular weight of polysaccharides can be measured by size exclusion chromatography (SEC) combined with a multi-angle laser scattering detector (MALLS).

[0052] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 sugar of the present invention has a weight-average molecular weight of 5 kDa to 5000 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 sugar has a weight-average molecular weight of 5 kDa to 2000 kDa.

[0053] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 50 kDa to 5000 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 50 kDa to 2000 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 50 kDa to 1000 kDa.

[0054] In one embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 polysaccharide has a weight-average molecular weight of 100 kDa to 5000 kDa. In another embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 polysaccharide has a weight-average molecular weight of 100 kDa to 2000 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 polysaccharide has a weight-average molecular weight of 100 kDa to 1000 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 polysaccharide has a weight-average molecular weight of 100 kDa to 500 kDa.

[0055] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 300 kDa to 5000 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 300 kDa to 2000 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 polysaccharide has a weight-average molecular weight of 300 kDa to 1000 kDa.

[0056] In one embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 polysaccharide has a weight-average molecular weight of 500 kDa to 3000 kDa. In another embodiment, the isolated polysaccharide has a weight-average molecular weight of 500 kDa to 2000 kDa. In yet another embodiment, the isolated polysaccharide has a weight-average molecular weight of 500 kDa to 1000 kDa.

[0057] Preferably, sugar sizing to a target molecular weight range is performed before conjugation to a carrier protein in order to generate a sugar conjugate exhibiting advantageous filtration properties, immunogenicity, and / or yield.

[0058] Advantageously, the size of purified Streptococcus pneumoniae serotype 21 sugars is reduced while retaining key structural features of the polysaccharide. Mechanical or chemical sizing can be used.

[0059] In one embodiment, the size of the purified Streptococcus pneumoniae (S. pneumoniae) serotype 21 capsule sugar is reduced by chemical hydrolysis. Chemical hydrolysis can be carried out using a weak acid (e.g., acetic acid, formic acid, propanoic acid). In one embodiment, chemical hydrolysis is carried out using formic acid. In one embodiment, chemical hydrolysis is carried out using propanoic acid. In a preferred embodiment, chemical hydrolysis is carried out using acetic acid. Chemical hydrolysis can also be carried out using a dilute strong acid (such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid, dilute nitric acid, or dilute perchloric acid). In one embodiment, chemical hydrolysis is carried out using dilute hydrochloric acid. In one embodiment, chemical hydrolysis is carried out using dilute sulfuric acid. In one embodiment, chemical hydrolysis is carried out using dilute phosphoric acid. In one embodiment, chemical hydrolysis is carried out using dilute nitric acid. In one embodiment, chemical hydrolysis is carried out using dilute perchloric acid.

[0060] The size of purified Streptococcus pneumoniae (S. pneumoniae) serotype 21 capsule sugars can also be reduced by mechanical homogenization. In one embodiment, the size of purified capsule sugars is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process flow through a sufficiently small volume channel. The shear rate can be increased by using a larger applied homogenization pressure and the exposure time can be increased by recirculating the feed flow through the homogenizer.

[0061] High-pressure homogenization processes may be suitable for reducing the size of purified capsule sugars while preserving the structural characteristics of the polysaccharides.

[0062] In a preferred embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 10 kDa to 1000 kDa. In a certain embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 50 kDa to 500 kDa. In a certain embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 50 kDa to 400 kDa. In a certain embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 50 kDa to 250 kDa.

[0063] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 100 kDa to 1000 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 100 kDa to 500 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 100 kDa to 400 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 100 kDa to 250 kDa.

[0064] In a preferred embodiment, the isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 capsule sugar is sized to a weight-average molecular weight of 150 kDa to 400 kDa.

[0065] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 250 kDa to 1000 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 250 kDa to 500 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 250 kDa to 400 kDa. In a preferred embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of 200 kDa to 800 kDa.

[0066] In one embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 250 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 300 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 350 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 400 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 450 kDa. In one embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 500 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 550 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 600 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 700 kDa. In yet another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 800 kDa. In one embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 900 kDa. In another embodiment, the isolated Streptococcus pneumoniae serotype 21 capsular sugar is sized to a weight-average molecular weight of approximately 1000 kDa.

[0067] In one embodiment, the isolated capsule sugar is not sized.

[0068] The isolated capsule sugars described above can be activated (e.g., chemically activated) to enable them to react, and then incorporated into sugar conjugates as further described herein.

[0069] 2. The present invention provides a Streptococcus pneumoniae serotype 21 glycoconjugate. For the purposes of this invention, the term “sugar conjugate” refers to a capsular sugar conjugated to a carrier protein via covalent or non-covalent bonds. In some embodiments, the capsular sugar is conjugated to the carrier protein via non-covalent bonds (e.g., rizavidin / biotin systems; see, e.g., WO2012155007, WO2020056202). Preferably, the capsular sugar is conjugated via covalent bonds. In one embodiment, the capsular sugar is directly conjugated to the carrier protein. In a second embodiment, the capsular sugar is conjugated to the carrier protein via a spacer / linker.

[0070] The present invention provides a sugar conjugate in which the sugars provided above are conjugated to a carrier protein. Accordingly, in one embodiment, the present invention provides a sugar conjugate comprising a sugar having the repeating units disclosed above, conjugated to a carrier protein.

[0071] In one embodiment, the present invention provides a sugar conjugate comprising a sugar having the repeating units disclosed above, conjugated to a carrier protein.

[0072] 2.1 Characteristics of the Streptococcus pneumoniae serotype 21 glycoconjugate of the present invention The isolated polysaccharides described above can be activated (e.g., chemically activated) to enable them to react (e.g., with a linker or directly with a carrier protein) and then incorporated into a sugar conjugate as further described herein.

[0073] Prior to activation, the size of the isolated polysaccharide can be reduced while retaining important structural features of the polysaccharide. Mechanical or chemical sizing can be used. In some embodiments, the size of the isolated polysaccharide is reduced by chemical hydrolysis. The size of the isolated polysaccharide can also be reduced by mechanical homogenization. In some embodiments, the size of the isolated polysaccharide is reduced by high-pressure homogenization. High-pressure homogenization achieves a high shear rate by pumping the process flow through a sufficiently small volume channel. The shear rate can be increased by using a larger applied homogenization pressure and the exposure time can be increased by recirculating the feed flow through the homogenizer.

[0074] The weight-average molecular weight (Mw) of the sugar before conjugation refers to the Mw before sugar activation (i.e., after the final sizing step, but before the sugar is reacted with the activator). In the context of this invention, the Mw of the sugar is not substantially altered by the activation step, and the Mw of the sugar incorporated into the conjugate is similar to the Mw of the sugar measured before activation.

[0075] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 50 kDa to 1,000 kDa of the polysaccharide before conjugation.

[0076] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 100 kDa to 600 kDa of the polysaccharide before conjugation.

[0077] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 100 kDa to 400 kDa of the polysaccharide before conjugation.

[0078] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 150 kDa to 300 kDa of the polysaccharide before conjugation.

[0079] In some embodiments, the serotype 21 glycoconjugate of the present invention has a weight-average molecular weight (Mw) of 250 kDa to 20,000 kDa.

[0080] In other embodiments, the serotype 21 glycoconjugate has a weight-average molecular weight (Mw) of 500 kDa to 15,000 kDa.

[0081] In other embodiments, the serotype 21 glycoconjugate has a weight-average molecular weight (Mw) of 500 kDa to 10,000 kDa.

[0082] In yet another embodiment, the serotype 21 glycoconjugate has a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa.

[0083] In a preferred embodiment, the serotype 21 glycoconjugate has a weight-average molecular weight (Mw) of 1,000 kDa to 5,000 kDa.

[0084] In preferred embodiments, the serotype 21 sugar conjugate of the present invention, prepared by click chemistry, has a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, more preferably 800 kDa to 7,000 kDa. In some embodiments, the carrier protein of the serotype 21 sugar conjugate is SCP.

[0085] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, has a weight-average molecular weight (Mw) of 2,000 kDa to 10,500 kDa. In a certain embodiment, the carrier protein of the serotype 21 sugar conjugate is CRM 197 That is the case.

[0086] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, and more preferably 750 kDa to 7,500 kDa, and more preferably 800 kDa to 7,000 kDa. In a certain embodiment, the carrier protein of the serotype 21 sugar conjugate is SCP.

[0087] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 100 kDa to 400 kDa of the polysaccharide before conjugation, and having a weight-average molecular weight (Mw) of 500 kDa to 15,000 kDa, more preferably 2,000 kDa to 10,500 kDa. In a certain embodiment, the carrier protein of the serotype 21 sugar conjugate is CRM 197 That is the case.

[0088] Serotype 21 polysaccharides are O-acetylated, with the total amount of O-acetylation being approximately one O-acetyl group per polysaccharide repeat unit. The degree of O-acetylation of a polysaccharide can be determined by any method known in the art, for example, by proton NMR or ion-HPLC analysis (see, for example, Lemercinier et al. (1996) Carbohydrate Research 296:83~96; Jones et al. (2002) J. Pharmaceutical and Biomedical Analysis 30:1233~1247; Hestrin, S. (1949) J. Biol. Chem. 180:249~261).

[0089] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar containing an average of at least 0.5 O-acetyl groups per sugar repeat unit. In a preferred embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar containing an average of at least 0.8 O-acetyl groups per sugar repeat unit.

[0090] In preferred embodiments, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar containing an average of about 0.8 to about 1 O-acetyl group per sugar repeat unit.

[0091] In one embodiment, the ratio of O-acetyl groups per sugar repeat unit in the sugar conjugate to O-acetyl groups per sugar repeat unit in the isolated polysaccharide is at least 0.6. In a preferred embodiment, the ratio of O-acetyl groups per sugar repeat unit in the sugar conjugate to O-acetyl groups per sugar repeat unit in the isolated polysaccharide is at least 0.9.

[0092] Another way to characterize the serotype 21 sugar conjugate of the present invention is to characterize the carrier protein (e.g., CRM) that becomes conjugated to the sugar, which can be characterized as the range (degree of conjugation) of the conjugated lysine. 197 This depends on the number of lysine residues in SCP, DT, or TT. Evidence of lysine modification of the carrier protein due to covalent bonding to polysaccharides can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of recovered lysine residues compared to the carrier protein starting material used to produce the conjugate material. In preferred embodiments, the degree of conjugation of the serotype 21 sugar conjugate of the present invention is 2 to 15. In some embodiments, the degree of conjugation of the serotype 21 sugar conjugate of the present invention is 2 to 10. In some embodiments, the degree of conjugation of the serotype 21 sugar conjugate of the present invention is 3 to 5. In some embodiments, the degree of conjugation of the serotype 21 sugar conjugate of the present invention is 2 to 6. In some embodiments, the degree of conjugation of the serotype 21 sugar conjugate of the present invention is 4 to 10.

[0093] The serotype 21 sugar conjugates of the present invention can also be characterized by the ratio (w / w) of the sugar to the carrier protein. In some embodiments, the ratio (w / w) of serotype 21 sugar in the sugar conjugate to the carrier protein is 0.5 to 3.0. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 0.5 to 2.0. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 0.5 to 1.5. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 0.8 to 1.2. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 0.5 to 1.0. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 1.0 to 1.5. In other embodiments, the ratio (w / w) of the sugar to the carrier protein is 1.0 to 2.0. In further embodiments, the ratio (w / w) of the sugar to the carrier protein is 0.8 to 1.2. In preferred embodiments, the ratio of serum type 21 sugars in the conjugate to the carrier protein is 0.7–1.1. In some such embodiments, the carrier protein is CRM 197 That is the case.

[0094] In a preferred embodiment, for the serotype 21 sugar conjugate of the present invention prepared by click chemistry, the ratio (w / w) of serotype 21 sugar to the carrier protein in the sugar conjugate is 0.5 to 1.1. In a certain embodiment, the carrier protein of the serotype 21 sugar conjugate is SCP.

[0095] In a preferred embodiment, for the serotype 21 sugar conjugate of the present invention prepared by direct reductive amination, the ratio (w / w) of serotype 21 sugar to the carrier protein in the sugar conjugate is 0.5 to 1.3. In one embodiment, the carrier protein of the serotype 21 sugar conjugate is CRM 197 That is the case.

[0096] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, and more preferably 800 kDa to 7,000 kDa, and a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.1. In a particular embodiment, the carrier protein of the serotype 21 sugar sugar conjugate is SCP.

[0097] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 100 kDa to 400 kDa, 500 kDa to 15,000 kDa, more preferably 2,000 kDa to 10,500 kDa, of the polysaccharide before conjugation, and a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.3. In a particular embodiment, the carrier protein of the serotype 21 sugar sugar conjugate is CRM 197 That is the case.

[0098] The serotype 21 sugar conjugates and immunogenic compositions of the present invention are not conjugated to a carrier protein, but nevertheless may contain free sugars present in the sugar conjugate composition. The free sugars may be non-covalently bound to the sugar conjugate (i.e., non-covalently bound, adsorbed, or captured in or with it). In a preferred embodiment, the serotype 21 sugar conjugate contains less than 50% free serotype 21 sugars compared to the total amount of serotype 21 sugars. In a preferred embodiment, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugars compared to the total amount of serotype 21 sugars. In a more preferred embodiment, the serotype 21 sugar conjugate contains less than 20% free serotype 21 sugars compared to the total amount of serotype 21 sugars. In a more preferred embodiment, the serotype 21 sugar conjugate contains less than 15% free serotype 21 sugars compared to the total amount of serotype 21 sugars.

[0099] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, having a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, more preferably 800 kDa to 7,000 kDa, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.1, and the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar. In a particular embodiment, the carrier protein of the serotype 21 sugar sugar conjugate is SCP.

[0100] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 100 kDa to 400 kDa, 500 kDa to 15,000 kDa, more preferably 2,000 kDa to 10,500 kDa of the polysaccharide before conjugation, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.3, and the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar. In a certain embodiment, the carrier protein of the serotype 21 sugar sugar conjugate is CRM 197 That is the case.

[0101] In a preferred embodiment, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, comprises a serotype 21 polysaccharide having a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, having a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, more preferably 800 kDa to 7,000 kDa, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.1, a degree of conjugation of the serotype 21 sugar conjugate of 2 to 10, and the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar. In a particular embodiment, the carrier protein of the serotype 21 sugar sugar conjugate is SCP.

[0102] In a preferred embodiment, the serotype 21 saccharide conjugate of the present invention prepared by direct reductive amination has a weight average molecular weight (Mw) of the polysaccharide before conjugation of 100 kDa to 400 kDa, and a weight average molecular weight (Mw) of 500 kDa to 15,000 kDa, more preferably 2,000 kDa to 10,500 kDa. The ratio (w / w) of the serotype 21 saccharide in the saccharide conjugate to the carrier protein is 0.5 to 1.3. The degree of conjugation of the serotype 21 saccharide conjugate is 2 to 10. The serotype 21 saccharide conjugate contains serotype 21 polysaccharide and contains less than about 25% free serotype 21 saccharide compared to the total amount of serotype 21 saccharide. In certain embodiments, the carrier protein of the saccharide conjugate of the serotype 21 saccharide is CRM 197 is.

[0103] Also, the serotype 21 saccharide conjugate can be characterized by its molecular size distribution (K d ). The relative molecular size distribution of the conjugate can be determined using a size exclusion chromatography medium (CL-4B). Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugate. Large molecules excluded from the small pores in the medium elute more rapidly than small molecules. A fraction collector is used to collect the column eluate. The fractions are tested by colorimetric analysis with a saccharide assay. For the determination of K d , the column is calibrated to establish the fraction (V0) where the molecule is completely excluded, (K d = 0), and the fraction (V i ) showing maximum retention, (K d = 1). The fraction (V e ) reaching a specific sample property is related to K d by the equation K e = (V i - V0) / (V d - V0).

[0104] In a preferred embodiment, at least 30% of the serotype 21 saccharide conjugate has a K value below or equal to 0.3 in the CL-4B column.d In a preferred embodiment, at least 40% of the sugar conjugate has a K value below or equal to 0.3 in the CL-4B column. d In a preferred embodiment, at least 60% of the serum type 21 sugar conjugates have a K value below or equal to 0.3 in the CL-4B column. d In a preferred embodiment, 50% to 80% of the serum type 21 sugar conjugates have a K value below or equal to 0.3 in the CL-4B column. d In a preferred embodiment, 65% to 80% of the serotype 21 sugar conjugates have a K value below or equal to 0.3 in the CL-4B column. d It holds.

[0105] In preferred embodiments, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, has a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, and more preferably 800 kDa to 7,000 kDa, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.1, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar, and 65% to 80% of the serotype 21 sugar conjugate has a K value below or equal to 0.3 in the CL-4B column. d It contains a serotype 21 polysaccharide having the following properties. In one embodiment, the carrier protein of the sugar conjugate of the serotype 21 sugar is SCP.

[0106] In preferred embodiments, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, has a weight-average molecular weight (Mw) of the polysaccharide before conjugation of 100 kDa to 400 kDa, more preferably 500 kDa to 15,000 kDa, and more preferably 2,000 kDa to 10,500 kDa, with a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.3, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar, and 65% to 80% of the serotype 21 sugar conjugate has a K value below or equal to 0.3 in the CL-4B column. d It contains a serotype 21 polysaccharide having the following characteristics. In one embodiment, the carrier protein of the sugar conjugate of the serotype 21 sugar is CRM 197 That is the case.

[0107] In preferred embodiments, the serotype 21 sugar conjugate of the present invention, prepared by Crick Chemistry, has a weight-average molecular weight (Mw) of 150 kDa to 400 kDa of the polysaccharide before conjugation, a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa, and more preferably 800 kDa to 7,000 kDa, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.1, a degree of conjugation of the serotype 21 sugar conjugate of 2 to 10, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar, and 65% to 80% of the serotype 21 sugar conjugate has a K value below or equal to 0.3 in the CL-4B column. d It contains a serotype 21 polysaccharide having the following properties. In one embodiment, the carrier protein of the sugar conjugate of the serotype 21 sugar is SCP.

[0108] In preferred embodiments, the serotype 21 sugar conjugate of the present invention, prepared by direct reductive amination, has a weight-average molecular weight (Mw) of the polysaccharide before conjugation of 100 kDa to 400 kDa, 500 kDa to 15,000 kDa, and more preferably 2,000 kDa to 10,500 kDa, a ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate of 0.5 to 1.3, a degree of conjugation of the serotype 21 sugar conjugate of 2 to 10, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar, and 65% to 80% of the serotype 21 sugar conjugate has a K value below or equal to 0.3 in the CL-4B column. d It contains a serotype 21 polysaccharide having the following characteristics. In one embodiment, the carrier protein of the sugar conjugate of the serotype 21 sugar is CRM 197 That is the case.

[0109] 2.2 Preparation method of the Streptococcus pneumoniae serotype 21 glycoconjugate of the present invention The serotype 21 glycoconjugate of the present invention can be prepared by any coupling technique known to those skilled in the art.

[0110] In one embodiment, the serotype 21 sugar is coupled to a carrier protein via a non-covalent bond (see, for example, WO2012155007 and WO2020056202).

[0111] In one embodiment, the serum type 21 sugar is conjugated via covalent bonding. In another embodiment, the capsular sugar is directly conjugated to the carrier protein. In a second embodiment, the capsular sugar is conjugated to the carrier protein via a spacer / linker.

[0112] In one embodiment, the serum type 21 sugar conjugate of the present invention is conjugated to a carrier protein via a linker, for example, a bifunctional linker. The linker may be heterodifunctional or homodifunctional, for example, having a reactive amino group and a reactive carboxylic acid group, two reactive amino groups or two reactive carboxylic acid groups. The linker may have, for example, 4 to 20, 4 to 12, or 5 to 10 carbon atoms.

[0113] A possible linker is adipic acid dihydrazide (ADH). Other linkers include β-propionamide (WO00 / 10599), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165;171~288), haloalkyl halides (US Patent No. 4,057,685), glycosidic bonds (US Patent No. 4,673,574, US Patent No. 4,808,700), hexanediamine, and 6-aminocaproic acid (US Patent No. 4,459,286).

[0114] In one embodiment, the serum type 21 glycoconjugate of the present invention is directly conjugated to a carrier protein (without a linker).

[0115] Generally, the following types of chemical groups on a protein carrier can be used for coupling / conjugation: 1) Amino group (e.g., via lysine). In one embodiment, this group is linked directly to a carboxyl group on a sugar, or to a carboxyl group on a linker using carbodiimide chemistry, for example, EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). In another embodiment, this group is linked directly to a hydroxyl group activated with CDAP or CNBr on a sugar, or to such a group on a linker; to a sugar or linker having an aldehyde group; or to a sugar or linker having a succinimide ester group. 2) Carboxyl group (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is linked directly to an amino group on a sugar, or to an amino group on a linker using carbodiimide chemistry, for example, using EDAC. 3) Sulfhydryl (e.g., via cysteine). In one embodiment, this group is linked to a bromo- or chloroacetylated sugar or linker using maleimide chemistry. In one embodiment, this group is activated / modified using bisdiazobenzidine. 4) A hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified using bisdiazobenzidine. 5) Imidazolyl group (e.g., via histidine). In one embodiment, this group is activated / modified using bisdiazobenzidine. 6) Guanidyl group (e.g., via arginine). 7) Indolyl group (e.g., via tryptophan).

[0116] On serum type 21 sugars, the following groups can generally be used for coupling: OH, COOH, or NH2. Aldehyde groups can be generated after various treatments known in the art, such as periodate, acid hydrolysis, and hydrogen peroxide.

[0117] In one embodiment, the serotype 21 sugar conjugate of the present invention is prepared using CDAP chemistry. In this embodiment, the serotype 21 sugar is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroboric acid (CDAP) to form a cyanate ester. Thus, the activated sugar can be coupled directly to an amino group on a carrier protein, or via a spacer (linker) group. For example, the spacer may be a cystamine or cysteamine for obtaining a thiolated polysaccharide that can be coupled to the carrier by a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidylbromoacetic acid (SBA;SIB), N-succinimidyl(4-iodoacetyl)aminobenzoic acid (SIAB), sulfosuccinimidyl(4-iodoacetyl)aminobenzoic acid (sulfo-SIAB), N-succinimidyliodoacetic acid (SIA), or succinimidyl 3-[bromoacetamide]propionic acid (SBAP)).

[0118] In preferred embodiments, the cyanate ester of the activated sugar is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivativeized sugar is conjugated to the carrier protein via a carboxyl group on the protein carrier using carbodiimide (e.g., EDAC or EDC) chemistry. Such conjugates are described, for example, in WO93 / 15760, WO95 / 08348 and WO96 / 129094.

[0119] In some embodiments, the serotype 21 sugar conjugates of the present invention are prepared using carbodiimide, hydrazide, active ester, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Many are described in International Patent Application Publication No. WO98 / 42721. The conjugation may also include a carbonyl linker that can be formed by reacting the free hydroxyl group of the sugar with CDI (see Bethell et al. (1979) J. Biol. Chem. 254:2572-2574; Hearn et al. (1981) J. Chromatogr. 218:509-518), and then reacting it with a protein to form a carbamate bond. This may include reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling of the CDI carbamate intermediate with an amino group on a protein.

[0120] Direct reductive amination In one embodiment, the serotype 21 sugar conjugate of the present invention is prepared by direct reductive amination (see, for example, U.S. Patents 4,365,170, 4,673,574, WO2006 / 110381, WO2008 / 079653, WO2008 / 143709, WO2008 / 079732, WO2011 / 110531, WO2012 / 119972, WO2015110941, WO2015110940, WO2018 / 144439, and WO2018 / 156491).

[0121] According to the present invention, reductive amination is a two-step process: (1) oxidation (activation) of the purified sugar of serum type 21, and (2) the activated sugar and carrier protein (e.g., CRM) for forming a sugar conjugate. 197 Includes reduction of TT or SCP.

[0122] As described above, serotype 21 sugars can be sized to a target molecular weight (MW) range before oxidation. Therefore, in some embodiments, isolated polysaccharides are sized before oxidation.

[0123] In one embodiment, the serum type 21 sugar of the present invention is used (a) A step of reacting the serum type 21 sugar with an oxidizing agent; (b) a step of mixing the activated sugar from step (a) with the carrier protein; and (c) A step in which the mixed, activated sugar and carrier protein are reacted with a reducing agent to form a sugar conjugate. The carrier protein is conjugated by a process that includes [a specific component / method].

[0124] In one embodiment, the serum type 21 sugar of the present invention is used (a) A step of reacting the serum type 21 sugar with an oxidizing agent; (a') A step to quench the oxidation reaction by adding a quenching agent; (b) A step of mixing the activated sugar from step (a') with the carrier protein; and (c) A step in which the mixed, activated sugar and carrier protein are reacted with a reducing agent to form a sugar conjugate. The carrier protein is conjugated by a process that includes [a specific component / method].

[0125] After the oxidation step (a), the sugar is said to be activated and is referred to as "activated sugar."

[0126] In some embodiments, the oxidizing agent is any oxidizing agent that oxidizes terminal hydroxyl groups to an aldehyde. In some embodiments, the oxidizing agent is a periodate. For the purposes of the present invention, the term "periodate" includes both periodates and periodic acid; this term also includes metaperiodic acid (IO4). - ) and orthoperiodate (IO6 5-This also includes both ) and various salts of periodic acid (e.g., sodium periodate and potassium periodate).

[0127] In one embodiment, the oxidizing agent is a periodate in the presence of a divalent cation (see WO2008 / 143709).

[0128] In one embodiment, the oxidizing agent is periodic acid. In another embodiment, the oxidizing agent is periodic acid in the presence of a divalent cation. In another embodiment, the oxidizing agent is Mg 2+ It is periodic acid in the presence of . In one embodiment, the oxidizing agent is Ca 2+ This is periodic acid in the presence of [a specific substance]. In some embodiments, the oxidizing agent is orthoperiodate.

[0129] In a preferred embodiment, the oxidizing agent is sodium periodate. In a certain embodiment, the periodate used for oxidation is metaperiodate. In a certain embodiment, the periodate used for oxidation is sodium metaperiodate.

[0130] When polysaccharides react with periodates, the periodates oxidize adjacent hydroxyl groups to form carbonyl or aldehyde groups, causing cleavage of the CC bond. Therefore, the term "reacting polysaccharides with periodates" includes the oxidation of adjacent hydroxyl groups by periodates.

[0131] In one embodiment, step a) includes reacting the polysaccharide with 0.01 to 2 molar equivalents of periodate. In one embodiment, step a) includes reacting the polysaccharide with 0.1 to 1.0 molar equivalents of periodate. In one embodiment, step a) includes reacting the polysaccharide with 0.1 to 0.5 molar equivalents of periodate.

[0132] In one embodiment, the oxidizing agent is a mixture of a stable nitroxyl radical compound and the oxidizing agent (see WO2014097099).

[0133] In one embodiment, the stable nitroxyl radical compound is a molecule having a TEMPO or PROXYL(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. Preferably, the molecule has the ability to selectively oxidize a primary alcohol in the presence of an oxidizing agent to produce an aldehyde group without affecting the secondary hydroxyl group. More preferably, the molecule has the ability to selectively oxidize a primary alcohol in the presence of an oxidizing agent to produce an aldehyde group without excessive oxidation to the carboxyl group. In one embodiment, the stable nitroxyl radical compound is TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamide)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamide)-TEMPO, or 4-amino-TEMPO, 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO. In one embodiment, the stable nitroxyl radical compound is selected from the group consisting of TEMPO, 2,2,6,6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinooxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetamide)-TEMPO free radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetamide)-TEMPO, 4-amino-TEMPO, and 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl. Preferably, the stable nitroxyl radical compound is TEMPO.In a further embodiment, the stable nitroxyl radical compound is 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, methyl 5-DOXYL-stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL. In a further embodiment, the stable nitroxyl radical compound is selected from the group consisting of 3β-DOXYL-5α-cholestane, 5-DOXYL-stearic acid, 16-DOXYL-stearic acid, methyl 5-DOXYL-stearate, 3-(aminomethyl)-PROXYL, 3-carbamoyl-PROXYL, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, 3-carboxy-PROXYL, or 3-cyano-PROXYL.

[0134] In one embodiment, the oxidizing agent is a molecule having an N-halo moiety. Preferably, the molecule has the ability to selectively oxidize a primary alcohol in the presence of a nitroxyl radical compound. In one embodiment, the oxidizing agent is N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, diiodoisocyanuric acid, or 1,3,5-triiodo-1,3,5-triazinan-2,4,6-trione. In one embodiment, the oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinan-2,4,6-trione. Preferably, the oxidizing agent is N-chlorosuccinimide.

[0135] In one aspect, the stable nitroxyl radical compound is 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO), and the oxidizing agent is N-chlorosuccinimide (NCS).

[0136] In one embodiment, the quenching agent in step a') is selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfites, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites or phosphorous acid.

[0137] In one embodiment, the quenching agent is of formula (I):

[0138]

Chemical formula

[0139] In one embodiment, the quenching agent is selected from the sodium and potassium salts of sulfites, bisulfites, dithionites, metabisulfites, thiosulfates, phosphites, hypophosphites or phosphorous acid.

[0140] In one embodiment, the quenching agent is an amino acid. In such an embodiment, the amino acid can be selected from serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0141] In one embodiment, the quenching agent is a sulfite such as bisulfite, dithionite, metabisulfite, thiosulfate.

[0142] In one embodiment, the quenching agent is a compound containing two adjacent hydroxyl groups (adjacent diols), that is, two hydroxyl groups covalently bonded to two adjacent carbon atoms.

[0143] Preferably, the quenching agent is of formula (II):

[0144] [ka] (In the formula, R 1 and R 2 (Each of these is independently selected from H, methyl, ethyl, propyl, or isopropyl.) It is a compound of [the compound].

[0145] In preferred embodiments, the quenching agent is glycerol, ethylene glycol, propane-1,2-diol, butane-1,2-diol or butane-2,3-diol, or ascorbic acid. In even more preferred embodiments, the quenching agent is butane-2,3-diol.

[0146] In a preferred embodiment, the degree of oxidation (DO) of the activated serotype 21 sugar is 2 to 30. In a certain embodiment, the degree of oxidation (DO) of the activated serotype 21 polysaccharide is 10 to 25.

[0147] In a very preferred embodiment, the degree of oxidation of the activated serum type 21 sugar is 10 to 21.

[0148] In one embodiment, the activated sugar and carrier protein are freeze-dried before step b).

[0149] In one embodiment, the initial input ratio (weight / weight) of the activated serotype 21 sugar to the carrier protein in step b) is 4:1 to 0.1:1. In another embodiment, the initial input ratio (weight / weight) of the activated serotype 21 sugar to the carrier protein in step b) is 1.5:1 to 0.5:1.

[0150] In one embodiment, the reduction reaction (c) is carried out in an aqueous solvent. In another embodiment, the reduction reaction (c) is carried out in an aprotic solvent.

[0151] In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0152] In one embodiment, the reduction reaction (c) is carried out in a solution essentially consisting of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in a solution essentially consisting of dimethylformamide (DMF). In one embodiment, the reduction reaction (c) is carried out in a solution essentially consisting of dimethyl sulfoxide (DMSO).

[0153] In one embodiment, the reduction reaction (c) is carried out in a DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent.

[0154] In one embodiment, the reducing agent is sodium borohydride cyanohydride, sodium borohydride triacetoxyhydride, Bronsted or sodium borohydride or zinc in the presence of Lewis acid, pyridineborane, 2-picolinborane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe iThe reducing agent is an amine borane such as PrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridineborane (PEMB). In some embodiments, the reducing agent is sodium triacetoxyborohydride. In preferred embodiments, the reducing agent is sodium cyanoborohydride. In some embodiments, the reducing agent is sodium cyanoborohydride in the presence of nickel (see WO2018144439).

[0155] In one embodiment, 0.2 to 20 molar equivalents of reducing agent are used in step c). In one embodiment, 0.5 to 10 molar equivalents of reducing agent are used in step c). In one embodiment, 1.0 to 5 molar equivalents of reducing agent are used in step c).

[0156] At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH4). In one embodiment, capping is achieved by mixing the product of step c) with 1 to 20 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by mixing the product of step c) with 1 to 10 molar equivalents of sodium borohydride. In one embodiment, capping is achieved by mixing the product of step c) with 1 to 5 molar equivalents of sodium borohydride.

[0157] Following conjugation to a carrier protein, the glycoconjugate can be purified (enriched in terms of the amount of glycoprotein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Thus, in one embodiment, the process for producing the glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its production.

[0158] CDI and / or CDT chemistry In certain embodiments, the serotype 21 saccharide conjugate of the invention is prepared using the CDI and / or CDT chemistry disclosed in WO2022249107.

[0159] CDI and / or CDT chemistry involves two steps: (1) reacting a serotype 21 saccharide with CDI and / or CDT in an aprotic solvent to produce an activated saccharide (activation), and (2) reacting the activated saccharide with a carrier protein (e.g., CRM 197 , TT or SCP) to form a saccharide conjugate.

[0160] In certain embodiments, the activator in step (1) is 1,1'-carbonyldiimidazole (CDI). In certain embodiments, the activator in step (1) is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT).

[0161] As described above, sizing of the serotype 21 saccharide to the target molecular weight (MW) range can be performed prior to oxidation using CDI and / or CDT.

[0162] Thus, in certain embodiments, the serotype 21 saccharide is sized prior to activation with CDI. In certain embodiments, the isolated polysaccharide is sized prior to activation with CDT. In certain embodiments, the serotype 21 saccharide is sized to any of the target molecular weight (MW) ranges defined above.

[0163] Thus, in certain embodiments, the serotype 21 saccharide is (a) reacting the isolated polysaccharide with CDI and / or CDT in an aprotic solvent; (b) reacting the activated polysaccharide of step (a) with a carrier protein in an aprotic solvent to form a saccharide conjugate by a process comprising conjugating to a carrier protein.

[0164] After the oxidation step (a), the polysaccharide is said to be activated and is referred to as "activated polysaccharide."

[0165] In one embodiment, step a) includes reacting a serotype 21 sugar with CDI.

[0166] In one embodiment, step a) includes reacting the serotype 21 sugar with CDI in an amount of 0.5 to 10 molar equivalents relative to the amount of serotype 21 sugar present in the reaction mixture.

[0167] In one embodiment, step a) includes reacting a serotype 21 sugar with CDT.

[0168] In one embodiment, step a) includes reacting the serotype 21 sugar with CDT in an amount of 0.5 to 10 molar equivalents relative to the amount of serotype 21 sugar present in the reaction mixture.

[0169] In one embodiment, the activation reaction a) is carried out in the presence of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA). In another embodiment, the activation reaction a) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0170] In one embodiment, the activation reaction a) is carried out in a solution essentially consisting of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

[0171] In one embodiment, the conjugation reaction b) is carried out in the presence of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA).

[0172] In one embodiment, the conjugation reaction b) is carried out in the presence of dimethyl sulfoxide (DMSO).

[0173] In one embodiment, the conjugation reaction b) is carried out in a solution essentially consisting of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).

[0174] In one embodiment, a weak organic base may be added to the reaction mixture after the activation reaction a) but before the conjugation reaction b). The weak organic base may be added before or after the carrier protein is introduced to the reaction mixture. Thus, in one embodiment, the weak organic base is added to the reaction mixture before the carrier protein is introduced. In another embodiment, the weak organic base is added to the reaction mixture after the carrier protein has been introduced. The weak organic base can be selected from alkanamines, imidazoles, triazoles, pyridines, histidines, and guanidines. Examples of alkanamines include alkyl primary amines such as methylamine, ethylamine, propylamine, and isopropylamine; alkyl secondary amines such as dimethylamine, diethylamine, dipropylamine, and diisopropylamine; and alkyl tertiary amines such as trimethylamine, triethylamine, triisopropylamine, and di-N,N'-isopropylethylamine. In one embodiment, the weak organic base is an alkaneamine. In one embodiment, the weak organic base is an imidazole. In one embodiment, the weak organic base is a triazole. In some embodiments, the weak organic base is pyridine. In some embodiments, the weak organic base is histidine. In some embodiments, the weak organic base is guanidine.

[0175] In one embodiment, after the conjugation reaction b), the unconjugated reaction sites of the activated polysaccharide are hydrolyzed. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous solution to the conjugation solution. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to about 3.0 to about 10.0. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to about 7.0 to about 10.0. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to about 3.0 to about 7.0. In one embodiment, the unconjugated reaction sites are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to about 4.0. In one embodiment, the reaction sites that were not conjugated are hydrolyzed by adding an aqueous buffer solution to the conjugation solution and adjusting the pH to about 9.0.

[0176] eTEC Chemistry In one embodiment, the serotype 21 glycoconjugate of the present invention is prepared by eTEC chemistry as disclosed in WO2014027302.

[0177] The eTEC spacer contains seven linear atoms (i.e., -C(O)NH(CH2)2SCH2C(O)-) and provides stable thioether and amide bonds between the sugar and the carrier protein. The synthesis of the eTEC-bound sugar conjugate involves the reaction of the activated hydroxyl group of the sugar with the amino group of a thioalkylamine reagent, such as cystamine or cysteineamine or a salt thereof, to form a carbamate bond to the sugar for providing the thiolated sugar. The generation of one or more free sulfhydryl groups is achieved by reaction with a reducing agent for providing the activated thiolated sugar. The reaction of the free sulfhydryl group of the activated thiolated sugar with an activated carrier protein having one or more α-haloacetamide groups on the amine-containing residue generates a thioether bond to form a conjugate, and the carrier protein binds to the eTEC spacer via an amide bond.

[0178] Therefore, in one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0179] In one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the sugar is covalently linked to the eTEC spacer via a carbamate bond, and the carrier protein is covalently linked to the eTEC spacer via an amide bond.

[0180] The eTEC-linked sugar conjugate of the present invention is defined by general formula (III):

[0181] [ka] (In the formula, (sugar) represents serotype 21 sugar.) It can be represented by [this].

[0182] Equation (III) is a schematic diagram of the sugar conjugate of the present invention. It should be understood that there is not just one linkage between the sugar and the carrier protein. Rather, individual carrier protein (CP) molecules may be linked to more than one serotype 21 sugar molecules, and individual sugar molecules may be linked to more than one individual carrier protein (CP) molecules. Furthermore, most sugar repeat units remain unmodified, and the covalent linkage between the carrier protein and the sugar is limited to a small number of sugar repeat units.

[0183] Click Chemistry In one embodiment, the serotype 21 glycoconjugate of the present invention is prepared by click chemistry (see, for example, PCT / IB2023 / 050202).

[0184] Therefore, in one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and general formula (IV):

[0185] [ka] (In the formula, X is CH2(CH2) n’ (CH2CH2O) m CH2CH2, NHCO(CH2) n’ NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ and O(CH2CH2O) m Selected from the group consisting of CH2CH2, n' is selected from 1 to 10, m is selected from 1 to 4, and X' is CH2O(CH2) n’’ CH2C=O, CH2O(CH2CH2O) m’ (CH2) n’’ (Selected from the group consisting of CH2C=O, n'' selected from 0 to 10, m' selected from 0 to 4, the structure in square brackets represents the repeating unit of serotype 21 sugar, and n represents the number of repeating units.) It holds.

[0186] In certain embodiments, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is CH2(CH2) n’ n' is 2 and X' is CH2O(CH2) n’’ CH2C=O, and n'' is 1. This study targets serotype 21 glycoconjugates that possess the following characteristics:

[0187] In certain embodiments, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and general formula (V):

[0188] [ka] (In the formula, the structure in square brackets represents the repeating unit of serotype 21-saccharide, and n represents the number of repeating units.) This relates to a serotype 21 glycoconjugate having [specific characteristics].

[0189] Formulas (IV) and (V) are schematic diagrams of the sugar conjugates of the present invention. It should be understood that linkage does not exist for all sugar repeating units (structures in square brackets). Rather, most sugar repeating units remain unmodified, and covalent linkage between the carrier protein and the sugar is limited to a small number of sugar repeating units. Furthermore, individual carrier protein (CP) molecules may be linked to more than one sugar molecule, and individual sugar molecules may be linked to more than one individual carrier protein (CP) molecule. The structures in square brackets represent the repeating units of serotype 21 sugars.

[0190] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is CH2(CH2) n’ n' is selected from 1 to 10, and X' is CH2O(CH2) n’’The present invention provides a serum type 21 glycoconjugate having CH2C=O and n'' 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In a further embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 1 and n'' is 4. In another embodiment, n' is 2 and n'' is 4. In yet another embodiment, n' is 3 and n'' is 4. In a further embodiment, n' is 4 and n'' is 4. In a further embodiment, n' is 5 and n'' is 4.In a further embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In a further embodiment, n' is 4 and n'' is 5. In a further embodiment, n' is 5 and n'' is 5. In a further embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In a further embodiment, n' is 4 and n'' is 6. In a further embodiment, n' is 5 and n'' is 6. In a further embodiment, n' is 6 and n'' is 6.

[0191] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is CH2(CH2) n’ And n' is selected from 1 to 10, CH2O(CH2CH2O) m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0192] 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 another embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0193] In a particular embodiment, n' is 1, m' is 0, and n'' is 0. In another embodiment, n' is 1, m' is 1, and n'' is 0. In yet another embodiment, n' is 1, m' is 2, and n'' is 0. In yet another embodiment, n' is 1, m' is 3, and n'' is 0.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is (CH2CH2O)m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ The present invention provides a serum type 21 glycoconjugate having CH2C=O and n'' 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0224] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is (CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0225] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0226] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by general formula (IV) (wherein X is NHCO(CH2) n’ n' is selected from 1 to 10, and X' is CH2O(CH2) n’’The present invention provides a serum type 21 glycoconjugate having CH2C=O and n'' 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In a further embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 1 and n'' is 4. In another embodiment, n' is 2 and n'' is 4. In yet another embodiment, n' is 3 and n'' is 4. In a further embodiment, n' is 4 and n'' is 4. In a further embodiment, n' is 5 and n'' is 4.In a further embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In a further embodiment, n' is 4 and n'' is 5. In a further embodiment, n' is 5 and n'' is 5. In a further embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In a further embodiment, n' is 4 and n'' is 6. In a further embodiment, n' is 5 and n'' is 6. In a further embodiment, n' is 6 and n'' is 6.

[0251] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by general formula (IV) (wherein X is NHCO(CH2) n’ Here, n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0252] 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 another embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0253] In a particular embodiment, n' is 1, m' is 0, and n'' is 0. In another embodiment, n' is 1, m' is 1, and n'' is 0. In yet another embodiment, n' is 1, m' is 2, and n'' is 0. In yet another embodiment, n' is 1, m' is 3, and n'' is 0.

[0254] 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by general formula (IV) (wherein X is NHCO(CH2CH2O)m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ The present invention provides a serum type 21 glycoconjugate having CH2C=O and n'' 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0284] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by general formula (IV) (wherein X is NHCO(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0285] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0286] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (IV) (wherein X is CH2(CH2) n’ n' is selected from 1 to 10, and X' is CH2O(CH2) n’’ We provide a serotype 21 glycoconjugate having CH2C=O and n'' selected from 0 to 10.

[0311] 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 4. In yet another embodiment, n' is 5 and n'' is 4. In yet another embodiment, n' is 6 and n'' is 4.In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet another embodiment, n' is 4 and n'' is 5. In yet another embodiment, n' is 5 and n'' is 5. In yet another embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet another embodiment, n' is 4 and n'' is 6. In yet another embodiment, n' is 5 and n'' is 6. In yet another embodiment, n' is 6 and n'' is 6.

[0312] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is of general formula (IV) (wherein X is OCH2(CH2) n’ Here, n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0313] 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 another embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0314] In a particular embodiment, n' is 1, m' is 0, and n'' is 0. In another embodiment, n' is 1, m' is 1, and n'' is 0. In yet another embodiment, n' is 1, m' is 2, and n'' is 0. In yet another embodiment, n' is 1, m' is 3, and n'' is 0.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by the general formula (IV) (wherein X is O(CH2CH2O)m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ We provide a serotype 21 glycoconjugate having CH2C=O and n'' selected from 0 to 10.

[0345] 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0346] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and is represented by the general formula (IV) (wherein X is O(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ We provide a serotype 21 sugar conjugate having CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0347] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0348] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0369] 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.

[0370] 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.

[0371] 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.

[0372] In a very preferred embodiment of the present invention, the serotype 21 glycoconjugate of the present invention is prepared using click chemistry. The present invention also relates to a method for preparing a serotype 21 glycoconjugate, as disclosed above herein.

[0373] In one embodiment, Crick Chemistry involves three steps: (a) reacting isolated serum type 21 sugars with a carbonate derivative and an azid linker in an aprotic solvent to produce activated azid sugars (sugar activation); (b) reacting a carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, where the NHS moiety reacts with an amino group to form an amide bond, thereby obtaining an alkyne-functionalized carrier protein (carrier protein activation); and (c) converting the activated azid sugar from step (a) into Cu +1 The process may also include a step in which the activated alkyne-carrier protein from step (b) is reacted with the alkyne-carrier protein by a mediated azide-alkynecycloaddition reaction to form a glycoconjugate.

[0374] After step (a), the sugar is said to be activated and is referred to herein as “activated sugar” or “activated azido sugar.”

[0375] After step (b), the carrier is said to be activated and is referred to as the "activated carrier."

[0376] As described above, sugar sizing to a target molecular weight (MW) range can be performed prior to activation (a). Therefore, in some embodiments, the isolated serotype 21 sugar is sized prior to activation with the carbonate derivative and azid linker. In some embodiments, the isolated serotype 21 sugar is sized to one of the target molecular weight (MW) ranges defined above. In some embodiments, the isolated serotype 21 sugar is not sized prior to activation with the carbonate derivative and azid linker.

[0377] In one embodiment, the carbonate derivative is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), N,N'-disuccinimidylcarbonate (DSC), and N-hydroxysuccinimidylchloroformate.

[0378] In one embodiment, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI). In another embodiment, the carbonate derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In one embodiment, the carbonate derivative is N,N'-disuccinimidylcarbonate (DSC). In yet another embodiment, the carbonate derivative is N-hydroxysuccinimidylchloroformate.

[0379] In one embodiment, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI) or 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In one embodiment, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI). Preferably, the carbonate derivative is N,N'-disuccinimidylcarbonate (DSC).

[0380] In one embodiment, the azid linker is given by formula (X): H2N-X-N3(X) (In the formula, X is CH2(CH2) n (CH2CH2O) mCH2CH2, NHCO(CH2) n , NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n and O(CH2CH2O) m selected from the group consisting of CH2CH2, where n is selected from 1 to 10 and m is selected from 1 to 4) is a compound of

[0381] In one embodiment, the azide linker is of formula (X) (where 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 yet another 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.

[0382] In one embodiment, the azide linker is of formula (X) (where 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.

[0383] In one embodiment, the azide linker is of formula (X) (where X is NHCO(CH2) nThe compound is (where 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 yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0384] In one embodiment, the azid linker is given by formula (X) (wherein X is NHCO(CH2CH2O) m The compound is CH2CH2 (where m is selected from 1 to 4). In some embodiments, m is selected from 1 to 3. In some embodiments, 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 yet another embodiment, m is 4.

[0385] In one embodiment, the azid linker is given by formula (X) (wherein X is OCH2(CH2) n The compound is (where 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 yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0386] In one embodiment, the azid linker is given by formula (X) (wherein X is O(CH2CH2O)m The compound is CH2CH2 (where m is selected from 1 to 4). In some embodiments, m is selected from 1 to 3. In some embodiments, 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 yet another embodiment, m is 4.

[0387] In one embodiment, the azid linker is given by formula (XI):

[0388] [ka] It is a compound of [the compound].

[0389] In a preferred embodiment, the azidolinker is 3-azidopropylamine.

[0390] In one embodiment, the drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a drug having an N-hydroxysuccinimide (NHS) moiety and a terminal alkyne.

[0391] 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.

[0392] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is of formula (XII):

[0393] [ka] (In the formula, X is CH2O(CH2) n CH2C=O and CH2O(CH2CH2O) m (CH2) n (Selected from the group consisting of CH2C=O, n is selected from 0 to 10, and m is selected from 0 to 4) It is a compound of [the compound].

[0394] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a compound of formula (XII) (where X is CH2O(CH2) n 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 particular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In a further embodiment, n is 4. In a further embodiment, n is 5. In a further embodiment, n is 6. In a further embodiment, n is 7. In a further embodiment, n is 8. In a further embodiment, n is 9. In a further embodiment, n is 10.

[0395] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a compound of formula (XII) (where X is CH2O(CH2CH2O) m (CH2) nIt 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 particular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another 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 particular embodiment, m is 1. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In yet another embodiment, m is 4.

[0396] In one embodiment, n is selected from 0 to 5 and m is selected from 0 to 3. In another embodiment, n is selected from 0 to 5 and m is selected from 0 to 2.

[0397] In one embodiment, n is selected from 0 to 4 and m is selected from 0 to 3. In another embodiment, n is selected from 0 to 4 and m is selected from 0 to 2.

[0398] In one embodiment, n is selected from 0 to 3 and m is selected from 0 to 3. In another embodiment, n is selected from 0 to 3 and m is selected from 0 to 2.

[0399] In one embodiment, n is selected from 0 to 2 and m is selected from 0 to 3. In another embodiment, n is selected from 0 to 2 and m is selected from 0 to 2.

[0400] In one embodiment, n is selected from 0 to 1 and m is selected from 0 to 3. In another embodiment, n is selected from 0 to 1 and m is selected from 0 to 2.

[0401] In one embodiment, n is 0 and m is 0. In another embodiment, n is 1 and m is 0. In another embodiment, n is 2 and m is 0. In another embodiment, n is 3 and m is 0. In another embodiment, n is 4 and m is 0. In another embodiment, n is 5 and m is 0. In another embodiment, n is 6 and m is 0. In another embodiment, n is 7 and m is 0. In another embodiment, n is 8 and m is 0. In another embodiment, n is 9 and m is 0. In another embodiment, n is 10 and m is 0.

[0402] In one embodiment, n is 0 and m is 1. In another embodiment, n is 1 and m is 1. In another embodiment, n is 2 and m is 1. In another embodiment, n is 3 and m is 1. In another embodiment, n is 4 and m is 1. In another embodiment, n is 5 and m is 1. In another embodiment, n is 6 and m is 1. In another embodiment, n is 7 and m is 1. In another embodiment, n is 8 and m is 1. In another embodiment, n is 9 and m is 1. In another embodiment, n is 10 and m is 1.

[0403] In one embodiment, n is 0 and m is 2. In another embodiment, n is 1 and m is 2. In another embodiment, n is 2 and m is 2. In another embodiment, n is 3 and m is 2. In another embodiment, n is 4 and m is 2. In another embodiment, n is 5 and m is 2. In another embodiment, n is 6 and m is 2. In another embodiment, n is 7 and m is 2. In another embodiment, n is 8 and m is 2. In another embodiment, n is 9 and m is 2. In another embodiment, n is 10 and m is 2.

[0404] In one embodiment, n is 0 and m is 3. In another embodiment, n is 1 and m is 3. In another embodiment, n is 2 and m is 3. In another embodiment, n is 3 and m is 3. In another embodiment, n is 4 and m is 3. In another embodiment, n is 5 and m is 3. In another embodiment, n is 6 and m is 3. In another embodiment, n is 7 and m is 3. In another embodiment, n is 8 and m is 3. In another embodiment, n is 9 and m is 3. In another embodiment, n is 10 and m is 3.

[0405] In one embodiment, n is 0 and m is 4. In another embodiment, n is 1 and m is 4. In another embodiment, n is 2 and m is 4. In another embodiment, n is 3 and m is 4. In another embodiment, n is 4 and m is 4. In another embodiment, n is 5 and m is 4. In another embodiment, n is 6 and m is 4. In another embodiment, n is 7 and m is 4. In another embodiment, n is 8 and m is 4. In another embodiment, n is 9 and m is 4. In another embodiment, n is 10 and m is 4.

[0406] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group is of formula (XIII):

[0407] [ka] It is a compound of [the compound].

[0408] In one embodiment, step a) includes reacting a sugar with a carbonate derivative, and then in an aprotic solvent, reacting the sugar activated by the carbonate derivative with an azid linker to produce an activated azid sugar.

[0409] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.01 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0410] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.1 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0411] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.5 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0412] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0413] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 2 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0414] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 5 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0415] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0416] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.5 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0417] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.01 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0418] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.1 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0419] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0420] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0421] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0422] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0423] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0424] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0425] In one embodiment, in step a), the isolated sugar is reacted with a carbonic acid derivative in an aprotic solvent.

[0426] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO).

[0427] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethylacetamide. In another embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of N-methyl-2-pyrrolidone. In yet another embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of hexamethylphosphoramide (HMPA).

[0428] In a preferred embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO).

[0429] In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO).

[0430] In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethylacetamide. In another embodiment, the isolated sugar is reacted with a carbonate derivative in N-methyl-2-pyrrolidone. In yet another embodiment, the isolated sugar is reacted with a carbonate derivative in hexamethylphosphoramide (HMPA).

[0431] In a preferred embodiment, the isolated sugar is reacted with CDI in dimethyl sulfoxide (DMSO). In a certain embodiment, the isolated sugar is reacted with CDI in anhydrous DMSO.

[0432] Surprisingly, it was found that side reactions could be avoided by reacting the isolated sugar with CDI in an environment with a humidity level of approximately 0.1% to 1% (v / v). Therefore, in one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.2% (v / v) water.

[0433] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.2% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.3% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.4% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.5% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.6% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.7% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.8% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.9% (v / v) water.

[0434] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.2% (v / v) water.

[0435] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.2% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.3% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.4% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.5% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.6% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.7% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing approximately 0.9% (v / v) water.

[0436] In one embodiment, the free carbon dioxide derivative is then quenched by adding water before the addition of the azid drinker. Water can activate the free CDI.

[0437] Therefore, in one embodiment, water is added after the activation of the carbonic acid derivative. In one embodiment, water is added to bring the total water content of the mixture to about 1% to about 10% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 1% to about 5% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 1% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 2% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 5% (v / v).

[0438] After reacting sugar with a carbonate derivative, the carbonate derivative is finally quenched with water, and then the sugar, activated by the carbonate derivative, is reacted with an azid linker.

[0439] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of azid linker that is 0.01 to 10 molar equivalents (molar equivalents of RU) relative to the amount of polysaccharide repeating units of the activated sugar.

[0440] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of azid linker that is 0.1 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0441] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of azid linker that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0442] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of azid linker that is 1 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0443] In the above embodiment, the carbonate derivative may be CDI. In another embodiment, the carbonate derivative is CDT. In a preferred embodiment, the carbonate derivative is DSC (N,N'-disuccinimidylcarbonate).

[0444] In one embodiment, the activation degree of the activated sugar after step a) is 1.0 to 100%. The activation degree of the azido sugar is defined as the percentage of repeating units linked to the azido linker.

[0445] In one embodiment, the activation degree of the activated sugar after step a) is 5-70%. In another embodiment, the activation degree of the activated sugar after step a) is 5-50%.

[0446] In a preferred embodiment, the activation degree of the activated sugar after step a) is 15-50%.

[0447] In another embodiment, the activation degree of the activated sugar after step a) is 10-40%.

[0448] In another embodiment, the activation degree of the activated sugar after step a) is 5-15%.

[0449] In another embodiment, the activation degree of the activated sugar after step a) is 15-35%.

[0450] In a preferred embodiment, the activation degree of the activated sugar after step a) is 15-50%.

[0451] In a very preferred embodiment, the activation degree of the activated sugar after step a) is 10-40%.

[0452] In one embodiment, the activation degree of the activated sugar after step a) is approximately 25%.

[0453] In one embodiment, the activation degree of the activated sugar after step a) is approximately 30%.

[0454] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of 0.1 to 10 molar equivalents relative to the lysine on the carrier.

[0455] In one embodiment, step b) includes reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of 0.5 to 10 molar equivalents relative to the lysine on the carrier.

[0456] In one embodiment, step b) includes reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, in an amount of 1 to 5 molar equivalents relative to the lysine on the carrier.

[0457] In one embodiment, step b) includes reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of 2 to 5 molar equivalents relative to the lysine on the carrier.

[0458] In one embodiment, step b) includes reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of 5 to 10 molar equivalents relative to the lysine on the carrier.

[0459] In one embodiment, step b) includes reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, in an amount of 1 to 5 molar equivalents relative to the lysine on the carrier.

[0460] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of about 10 molar equivalents relative to the lysine on the carrier.

[0461] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of about 5 molar equivalents relative to the lysine on the carrier.

[0462] In one embodiment, step b) includes reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an alkyne group, in an amount of about 2 molar equivalents relative to the lysine on the carrier.

[0463] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of about 1 molar equivalent relative to the lysine on the carrier.

[0464] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of about 0.5 molar equivalents relative to the lysine on the carrier.

[0465] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group in an amount of about 0.1 molar equivalents relative to the lysine on the carrier.

[0466] In one embodiment, the activation degree of the activated carrier after step b) is 1 to 50. The activation degree of the activated carrier is defined as the number of lysine residues in the carrier protein that become linked to the drug having an N-hydroxysuccinimide (NHS) moiety and an alkyne group.

[0467] In one embodiment, the carrier protein is a CRM containing 39 lysine residues. 197 In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 30. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 5 to 20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 9 to 18. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 8-11. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of the ) is 15-20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 5. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 6. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 7. In another embodiment, the activated carrier (CRM) after step b) 197The activation degree of ) is approximately 8. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 9. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 10. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 11. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 12. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 13. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 14. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 15. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 16. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 17. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 18. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 19. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 21. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 22. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 23. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 24. In another embodiment, the activated carrier (CRM) after step b) 197 The activation level of ) is approximately 25.

[0468] In one embodiment, the carrier protein is an SCP or a fragment thereof. In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 50. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 5 to 50. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 7 to 45. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 5 to 25. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 10 to 25. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 17 to 22. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is about 5. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is about 7. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is about 10. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 13. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 15. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 20. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 26. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 30. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 35. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 37. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 40. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 45. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 50.

[0469] In one embodiment, the carrier protein is TT or a fragment thereof. In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 30. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 5 to 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 7 to 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 10 to 20. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 5. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 7. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 10. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 12. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 15. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 20. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 30.

[0470] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer. In another embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst. In yet another embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as an oxidizing agent and a catalyst. In a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst and an ascorbate as an oxidizing agent. In one embodiment, THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine can be further added to protect the protein from side reactions. Therefore, in a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst and an ascorbate as an oxidizing agent, and the reaction mixture further comprises THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine.

[0471] In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is 0.1 to 3. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is 0.5 to 2. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is 0.6 to 1.5. In a preferred embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is 0.8 to 1. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is about 0.5. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is about 0.6. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 0.7. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 0.8. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 0.9. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.1. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.2. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.3. In another embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.4.In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.5. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.6. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.7. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.8. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 1.9. In one embodiment, the initial input ratio (weight / weight) of the activated azido sugar to the activated alkyne-carrier in step c) is approximately 2.

[0472] After the click conjugation reaction, unreacted azide groups may remain in the conjugate, and these can be capped using a suitable azide capping agent. Therefore, in one embodiment, after step c), the unreacted azide groups in the conjugate are capped using a suitable azide capping agent. In one embodiment, this azide capping agent is a drug having an alkyne group. In one embodiment, this azide capping agent is a drug having a terminal alkyne. In one embodiment, this azide capping agent is a drug having a cycloalkyne.

[0473] In one embodiment, the azide group capping agent is of formula (XIV):

[0474] [ka] (In the formula, X is (CH2) n (where n is selected from 1 to 15) It is a compound of [the compound].

[0475] In one embodiment, the azide group capping agent is propargyl alcohol.

[0476] Therefore, in one embodiment, after step (c), the process further includes the step of capping any unreacted azide groups remaining in the conjugate with an azide group capping agent.

[0477] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.05 to 20 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0478] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.1 to 15 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0479] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0480] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0481] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0482] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated sugar.

[0483] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 1 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0484] In one embodiment, capping of unreacted azide groups is carried out using a capping agent in an amount of 0.75 to 1.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0485] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is approximately 1 molar equivalent relative to the amount of polysaccharide repeating units of the activated sugar.

[0486] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is approximately 1.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0487] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is approximately 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0488] In one embodiment, capping of unreacted azide groups is carried out using an amount of capping agent that is approximately 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0489] After the click conjugation reaction, unreacted alkyne groups may remain 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.

[0490] In one embodiment, the alkyne group capping agent is of formula (XV): N3-X-OH(XV) (In the formula, X is (CH2) n (where n is selected from 1 to 15) It is a compound of [the compound].

[0491] In one embodiment, the alkyne group capping agent is 3-azido-1-propanol.

[0492] Therefore, in one embodiment, after step (c), the process further includes the step of capping any unreacted alkyne groups remaining in the conjugate with an alkyne group capping agent.

[0493] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.05 to 20 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0494] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.1 to 15 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0495] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.5 to 10 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0496] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.5 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0497] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0498] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 0.5 to 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated sugar.

[0499] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of 1 to 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0500] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1 to 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0501] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is 1.5 to 2.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0502] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is approximately 0.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0503] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is approximately 1 molar equivalent relative to the amount of polysaccharide repeat units of the activated sugar.

[0504] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is approximately 1.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0505] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is approximately 2 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0506] In one embodiment, capping of unreacted alkyne groups is carried out using an amount of capping agent that is approximately 2.5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0507] In one embodiment, capping of unreacted alkyne groups is carried out using a capping agent in an amount of about 5 molar equivalents relative to the amount of polysaccharide repeat units of the activated sugar.

[0508] Following conjugation to a carrier protein, the glycoconjugate can be purified (enriched in terms of the amount of glycoprotein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and deep filtration. Thus, in one embodiment, the process for producing the glycoconjugate of the present invention includes a step of purifying the glycoconjugate after its production.

[0509] In one embodiment, the present invention provides a serotype 21 glycoconjugate produced according to any of the methods disclosed herein.

[0510] Alternative click chemistry In one embodiment of the present invention, the serotype 21 glycoconjugate of the present invention is prepared, for example, by alternative click chemistry disclosed in application number PCT / IB2024 / 051122 (filed February 7, 2024).

[0511] Therefore, in one embodiment, the serotype 21 sugar conjugate of the present invention comprises a serotype 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and the general formula is (XVI):

[0512] [ka] (In the formula, X is CH2(CH2) n’ (CH2CH2O) m CH2CH2, NHCO(CH2) n’ NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n’ and O(CH2CH2O) m Selected from the group consisting of CH2CH2, n' is selected from 0 to 10, m is selected from 1 to 4, and X' is CH2(CH2) n’’ CH2O(CH2) n’’ CH2, CH2O (CH2CH2O) m’(CH2) n’’ (Selected from the group consisting of CH2, n'' is selected from 0 to 10, m' is selected from 0 to 4, the structure in square brackets represents the repeating unit of serotype 21-saccharide, and n represents the number of repeating units.) It has.

[0513] In certain embodiments, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is CH2(CH2) n’ Therefore, n' is 0, and X' is CH2(CH2) n’’ The target is serotype 21 glycoconjugate having n'' = 0.

[0514] In certain embodiments, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and general formula (XVII):

[0515] [ka] (In the formula, the structure in square brackets represents the repeating unit of serotype 21-saccharide, and n represents the number of repeating units.) This relates to a serotype 21 glycoconjugate having [specific characteristics].

[0516] Formulas (XVI) and (XVII) are schematic diagrams of the sugar conjugates of the present invention. It should be understood that linkage does not exist for all sugar repeating units (structures in square brackets). Rather, most sugar repeating units remain unmodified, and covalent linkage between the carrier protein and the sugar is for a small number of sugar repeating units. Furthermore, individual carrier protein (CP) molecules may be linked to more than one sugar molecule, and individual sugar molecules may be linked to more than one individual carrier protein (CP) molecule. The structures in square brackets represent the repeating units of serotype 21 sugars.

[0517] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is CH2(CH2) n’ n' is selected from 0 to 10, and X' is CH2(CH2) n’’The present invention provides a serotype 21 glycoconjugate having n' and n'' selected from 0 to 10. In one embodiment, n' is selected from 0 to 5 and n'' is selected from 0 to 10. In one embodiment, n' is selected from 0 to 5 and n'' is selected from 0 to 5. In one embodiment, n' is selected from 0 to 3 and n'' is selected from 0 to 3. In one embodiment, n' is selected from 0 to 2 and n'' is selected from 0 to 2. In a particular embodiment, n' is 0 and n'' is 0. 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another embodiment, n' is 6 and n'' is 0. In a particular embodiment, n' is a and n'' is 1. 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another embodiment, n' is 6 and n'' is 1. In a particular embodiment, n' is 0 and n'' is 2. 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another embodiment, n' is 6 and n'' is 2. In a particular embodiment, n' is 0 and n'' is 3. 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In yet another embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 0 and n'' is 4.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 yet another embodiment, n' is 4 and n'' is 4. In yet another embodiment, n' is 5 and n'' is 4. In yet another embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 0 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet another embodiment, n' is 4 and n'' is 5. In yet another embodiment, n' is 5 and n'' is 5. In yet another embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 0 and n'' is 6. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet another embodiment, n' is 4 and n'' is 6. In yet another embodiment, n' is 5 and n'' is 6. In yet another embodiment, n' is 6 and n'' is 6.

[0518] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is CH2(CH2) n’ n' is selected from 0 to 10, and X' is CH2O(CH2) n’’The present invention provides a serum type 21 glycoconjugate having CH2 and n'' selected from 0 to 10. In one embodiment, n' is selected from 0 to 5 and n'' is selected from 0 to 10. In one embodiment, n' is selected from 0 to 5 and n'' is selected from 0 to 5. In one embodiment, n' is selected from 0 to 3 and n'' is selected from 0 to 3. In one embodiment, n' is selected from 0 to 2 and n'' is selected from 0 to 2. In a particular embodiment, n' is 0 and n'' is 0. 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 a and n'' is 1. 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another embodiment, n' is 6 and n'' is 1. In a particular embodiment, n' is 0 and n'' is 2. 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another embodiment, n' is 6 and n'' is 2. In a particular embodiment, n' is 0 and n'' is 3. 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In yet another embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 0 and n'' is 4.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 yet another embodiment, n' is 4 and n'' is 4. In yet another embodiment, n' is 5 and n'' is 4. In yet another embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 0 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet another embodiment, n' is 4 and n'' is 5. In yet another embodiment, n' is 5 and n'' is 5. In yet another embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 0 and n'' is 6. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet another embodiment, n' is 4 and n'' is 6. In yet another embodiment, n' is 5 and n'' is 6. In yet another embodiment, n' is 6 and n'' is 6.

[0519] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is CH2(CH2) n’ n' is selected from 0 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0520] In one embodiment, n' is selected from 0 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 10. In another embodiment, n' is selected from 0 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 0 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 0 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1. In yet another embodiment, n' is selected from 0 to 1, m' is selected from 0 to 1, and n'' is selected from 0 to 1.

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

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

[0523] 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.

[0524] 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.

[0525] 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.

[0526] 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.

[0527] In certain embodiments, n' is 0, m' is 0, and n'' is 1. In certain embodiments, n' is 0, m' is 1, and n'' is 1. In certain embodiments, n' is 0, m' is 2, and n'' is 1. In certain embodiments, n' is 0, m' is 3, and n'' is 1.

[0528] 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.

[0529] 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.

[0530] 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.

[0531] 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.

[0532] 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.

[0533] In certain embodiments, n' is 0, m' is 0, and n'' is 2. In certain embodiments, n' is 0, m' is 1, and n'' is 2. In certain embodiments, n' is 0, m' is 2, and n'' is 2. In certain embodiments, n' is 0, m' is 3, and n'' is 2.

[0534] 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.

[0535] 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.

[0536] 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.

[0537] 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.

[0538] 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.

[0539] In certain embodiments, n' is 0, m' is 0, and n'' is 3. In certain embodiments, n' is 0, m' is 1, and n'' is 3. In certain embodiments, n' is 0, m' is 2, and n'' is 3. In certain embodiments, n' is 0, m' is 3, and n'' is 3.

[0540] 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.

[0541] 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.

[0542] 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.

[0543] 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.

[0544] 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.

[0545] In certain embodiments, n' is 0, m' is 0, and n'' is 4. In certain embodiments, n' is 0, m' is 1, and n'' is 4. In certain embodiments, n' is 0, m' is 2, and n'' is 4. In certain embodiments, n' is 0, m' is 3, and n'' is 4.

[0546] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0547] 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.

[0548] 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.

[0549] 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.

[0550] 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.

[0551] In certain embodiments, n' is 0, m' is 0, and n'' is 5. In certain embodiments, n' is 0, m' is 1, and n'' is 5. In certain embodiments, n' is 0, m' is 2, and n'' is 5. In certain embodiments, n' is 0, m' is 3, and n'' is 5.

[0552] 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.

[0553] 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.

[0554] 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.

[0555] 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.

[0556] 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.

[0557] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is (CH2CH2O) m The configuration is CH2CH2, where m is selected from 1 to 4, and X' is CH2(CH2). n’’The present invention provides a serum type 21 glycoconjugate having m = 1 and n'' = 0 (where n'' is selected from 0 to 10). In one embodiment, m = 1 to 3 and n'' = 0 to 10. In one embodiment, m = 1 to 3 and n'' = 0 to 5. In one embodiment, m = 1 to 2 and n'' = 0 to 3. In one embodiment, m = 1 to 2 and n'' = 0 to 2. In a particular embodiment, m = 1 and n'' = 0. In another embodiment, m = 2 and n'' = 0. In yet another embodiment, m = 3 and n'' = 0. In a further embodiment, m = 4 and n'' = 0. In a particular embodiment, m = 1 and n'' = 1. In another embodiment, m = 2 and n'' = 1. In yet another embodiment, m = 3 and n'' = 1. In a further embodiment, m = 4 and n'' = 1. In a particular embodiment, m = 1 and n'' = 2. In another embodiment, m is 2 and n'' is 2. In yet another embodiment, m is 3 and n'' is 2. In yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0558] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is (CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’The present invention provides a serum type 21 glycoconjugate having CH2 and n'' 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0559] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is (CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0560] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0561] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0562] 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.

[0563] 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.

[0564] 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.

[0565] 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.

[0566] 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.

[0567] 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.

[0568] 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.

[0569] 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.

[0570] 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.

[0571] 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.

[0572] 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.

[0573] 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.

[0574] 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.

[0575] 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.

[0576] 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.

[0577] 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.

[0578] 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.

[0579] 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.

[0580] 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.

[0581] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0582] 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.

[0583] 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.

[0584] 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.

[0585] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2) n’ n' is selected from 1 to 10, and X' is CH2(CH2) n’’The present invention provides a serotype 21 glycoconjugate having n' and n'' 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In a further embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 1 and n'' is 4. In another embodiment, n' is 2 and n'' is 4. In yet another embodiment, n' is 3 and n'' is 4. In a further embodiment, n' is 4 and n'' is 4. In a further embodiment, n' is 5 and n'' is 4.In a further embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In a further embodiment, n' is 4 and n'' is 5. In a further embodiment, n' is 5 and n'' is 5. In a further embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In a further embodiment, n' is 4 and n'' is 6. In a further embodiment, n' is 5 and n'' is 6. In a further embodiment, n' is 6 and n'' is 6.

[0586] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2) n’ n' is selected from 1 to 10, and X' is CH2O(CH2) n’’The present invention provides a serum type 21 glycoconjugate having CH2 and n'' 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 1. In yet another embodiment, n' is 5 and n'' is 1. In yet another 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 yet another embodiment, n' is 4 and n'' is 2. In yet another embodiment, n' is 5 and n'' is 2. In yet another 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 yet another embodiment, n' is 4 and n'' is 3. In yet another embodiment, n' is 5 and n'' is 3. In a further embodiment, n' is 6 and n'' is 3. In a particular embodiment, n' is 1 and n'' is 4. In another embodiment, n' is 2 and n'' is 4. In yet another embodiment, n' is 3 and n'' is 4. In a further embodiment, n' is 4 and n'' is 4. In a further embodiment, n' is 5 and n'' is 4.In a further embodiment, n' is 6 and n'' is 4. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In a further embodiment, n' is 4 and n'' is 5. In a further embodiment, n' is 5 and n'' is 5. In a further embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In a further embodiment, n' is 4 and n'' is 6. In a further embodiment, n' is 5 and n'' is 6. In a further embodiment, n' is 6 and n'' is 6.

[0587] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2) n’ Here, n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0588] 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 another embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0589] In a particular embodiment, n' is 1, m' is 0, and n'' is 0. In another embodiment, n' is 1, m' is 1, and n'' is 0. In yet another embodiment, n' is 1, m' is 2, and n'' is 0. In yet another embodiment, n' is 1, m' is 3, and n'' is 0.

[0590] 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.

[0591] 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.

[0592] 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.

[0593] 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.

[0594] 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.

[0595] 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.

[0596] 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.

[0597] 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.

[0598] 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.

[0599] 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.

[0600] 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.

[0601] 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.

[0602] 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.

[0603] 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.

[0604] 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.

[0605] 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.

[0606] 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.

[0607] 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.

[0608] 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.

[0609] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0610] 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.

[0611] 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.

[0612] 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.

[0613] 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.

[0614] 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.

[0615] 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.

[0616] 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.

[0617] 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.

[0618] 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.

[0619] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2CH2O)m The configuration is CH2CH2, where m is selected from 1 to 4, and X' is CH2(CH2). n’’ The present invention provides a serum type 21 glycoconjugate having m = 1 and n'' = 0 (where n'' is selected from 0 to 10). In one embodiment, m = 1 to 3 and n'' = 0 to 10. In one embodiment, m = 1 to 3 and n'' = 0 to 5. In one embodiment, m = 1 to 2 and n'' = 0 to 3. In one embodiment, m = 1 to 2 and n'' = 0 to 2. In a particular embodiment, m = 1 and n'' = 0. In another embodiment, m = 2 and n'' = 0. In yet another embodiment, m = 3 and n'' = 0. In a further embodiment, m = 4 and n'' = 0. In a particular embodiment, m = 1 and n'' = 1. In another embodiment, m = 2 and n'' = 1. In yet another embodiment, m = 3 and n'' = 1. In a further embodiment, m = 4 and n'' = 1. In a particular embodiment, m = 1 and n'' = 2. In another embodiment, m is 2 and n'' is 2. In yet another embodiment, m is 3 and n'' is 2. In yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0620] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’The present invention provides a serum type 21 glycoconjugate having CH2 and n'' 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0621] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is NHCO(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0622] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0623] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0624] 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.

[0625] 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.

[0626] 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.

[0627] 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.

[0628] 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.

[0629] 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.

[0630] 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.

[0631] 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.

[0632] 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.

[0633] 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.

[0634] 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.

[0635] 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.

[0636] 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.

[0637] 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.

[0638] 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.

[0639] 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.

[0640] 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.

[0641] 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.

[0642] 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.

[0643] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0644] 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.

[0645] 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.

[0646] 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.

[0647] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is OCH2(CH2) n’ n' is selected from 1 to 10, and X' is CH2(CH2) n’’ The present invention provides a serotype 21 glycoconjugate having n'' selected from 0 to 10.

[0648] 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 4. In yet another embodiment, n' is 5 and n'' is 4. In yet another embodiment, n' is 6 and n'' is 4.In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet another embodiment, n' is 4 and n'' is 5. In yet another embodiment, n' is 5 and n'' is 5. In yet another embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet another embodiment, n' is 4 and n'' is 6. In yet another embodiment, n' is 5 and n'' is 6. In yet another embodiment, n' is 6 and n'' is 6.

[0649] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is OCH2(CH2) n’ n' is selected from 1 to 10, and X' is CH2O(CH2) n’’ We provide a serum type 21 glycoconjugate having CH2 (where n'' is selected from 0 to 10).

[0650] 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 yet another embodiment, n' is 4 and n'' is 0. In yet another embodiment, n' is 5 and n'' is 0. In yet another 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 yet another embodiment, n' is 4 and n'' is 4. In yet another embodiment, n' is 5 and n'' is 4. In yet another embodiment, n' is 6 and n'' is 4.In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet another embodiment, n' is 4 and n'' is 5. In yet another embodiment, n' is 5 and n'' is 5. In yet another embodiment, n' is 6 and n'' is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet another embodiment, n' is 4 and n'' is 6. In yet another embodiment, n' is 5 and n'' is 6. In yet another embodiment, n' is 6 and n'' is 6.

[0651] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is OCH2(CH2) n’ Here, n' is selected from 1 to 10, and X' is CH2O(CH2CH2O) m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0652] 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 another embodiment, n' is selected from 1 to 5, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, n' is selected from 1 to 3, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, n' is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0653] In a particular embodiment, n' is 1, m' is 0, and n'' is 0. In another embodiment, n' is 1, m' is 1, and n'' is 0. In yet another embodiment, n' is 1, m' is 2, and n'' is 0. In yet another embodiment, n' is 1, m' is 3, and n'' is 0.

[0654] 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.

[0655] 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.

[0656] 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.

[0657] 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.

[0658] 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.

[0659] 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.

[0660] 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.

[0661] 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.

[0662] 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.

[0663] 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.

[0664] 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.

[0665] 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.

[0666] 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.

[0667] 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.

[0668] 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.

[0669] 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.

[0670] 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.

[0671] 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.

[0672] 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.

[0673] In certain embodiments, n' is 1, m' is 0, and n'' is 4. In certain embodiments, n' is 1, m' is 1, and n'' is 4. In certain embodiments, n' is 1, m' is 2, and n'' is 4. In certain embodiments, n' is 1, m' is 3, and n'' is 4.

[0674] 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.

[0675] 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.

[0676] 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.

[0677] 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.

[0678] 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.

[0679] 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.

[0680] 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.

[0681] 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.

[0682] 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.

[0683] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is O(CH2CH2O)m The configuration is CH2CH2, where m is selected from 1 to 4, and X' is CH2(CH2). n’’ The present invention provides a serotype 21 glycoconjugate having n'' selected from 0 to 10.

[0684] 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0685] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is O(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2). n’’ We provide a serum type 21 glycoconjugate having CH2 (where n'' is selected from 0 to 10).

[0686] 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another 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 yet another embodiment, m is 4 and n'' is 6.

[0687] In one embodiment, the present invention comprises a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, with the general formula (XVI) (wherein X is O(CH2CH2O) m The compound is CH2CH2, where m is selected from 1 to 4, and X' is CH2O(CH2CH2O). m’ (CH2) n’’ The present invention provides a serotype 21 glycoconjugate having CH2, where n'' is selected from 0 to 10 and m' is selected from 0 to 4.

[0688] 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 another embodiment, m is selected from 1 to 2, m' is selected from 0 to 4, and n'' is selected from 0 to 5. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 3. In yet another embodiment, m is selected from 1 to 2, m' is selected from 0 to 2, and n'' is selected from 0 to 1.

[0689] In a particular 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 yet another embodiment, m is 1, m' is 2, and n'' is 0. In yet another embodiment, m is 1, m' is 3, and n'' is 0.

[0690] 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.

[0691] 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.

[0692] 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.

[0693] 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.

[0694] 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.

[0695] 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.

[0696] 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.

[0697] 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.

[0698] 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.

[0699] 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.

[0700] 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.

[0701] 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.

[0702] 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.

[0703] 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.

[0704] 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.

[0705] 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.

[0706] 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.

[0707] 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.

[0708] 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.

[0709] In a particular embodiment, m is 1, m' is 0, and n'' is 5. In a particular embodiment, m is 1, m' is 1, and n'' is 5. In a particular embodiment, m is 1, m' is 2, and n'' is 5. In a particular embodiment, m is 1, m' is 3, and n'' is 5.

[0710] 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.

[0711] 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.

[0712] 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.

[0713] In one embodiment of the present invention, the serotype 21 glycoconjugate of the present invention is prepared using the alternative click chemistry described in this section. The present invention also relates to a method for preparing a serotype 21 glycoconjugate, as disclosed above herein.

[0714] In one embodiment, Crick Chemistry performs three steps: (a) in an aprotic solvent, the isolated serum type 21 sugar is reacted with a carbonate derivative and an alkyne linker to produce an activated alkynyl sugar (sugar activation); (b) the carrier protein is reacted with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group, where the NHS moiety reacts with an amino group to form an amide bond, thereby obtaining an azide-functionalized carrier protein (carrier protein activation); (c) the activated alkynyl sugar from step (a) is converted into Cu +1 The process may also include a step in which the activated azide-carrier protein from step (b) is reacted with the azide-alkynecycloaddition protein by a mediated azide-alkynecycloaddition reaction to form a glycoconjugate.

[0715] After step (a), the sugar is said to be activated and is referred to herein as “activated sugar” or “activated alkynyl sugar”.

[0716] After step (b), the carrier is said to be activated and is referred to as the "activated carrier" or "activated azide carrier."

[0717] As described above, serotype 21 sugars can be sized to a target molecular weight (MW) range prior to activation (a). Therefore, in some embodiments, isolated serotype 21 sugars are sized before activation with carbonate derivatives and alkyne linkers. In some embodiments, isolated serotype 21 sugars are sized to any of the target molecular weight (MW) ranges defined above. In some embodiments, isolated serotype 21 sugars are not sized before activation with carbonate derivatives and alkyne linkers.

[0718] In one embodiment, the carbonate derivative is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidylcarbonate (DSC), and N-hydroxysuccinimidylchloroformate.

[0719] In one embodiment, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI). In another embodiment, the carbonate derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In yet another embodiment, the carbonate derivative is disuccinimidylcarbonate (DSC). In yet another embodiment, the carbonate derivative is N-hydroxysuccinimidylchloroformate.

[0720] In some embodiments, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI) or 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In some embodiments, the carbonate derivative is 1,1'-carbonyldiimidazole (CDI). In some embodiments, the carbonate derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). Preferably, the carbonate derivative is N,N'-disuccinimidylcarbonate (DSC).

[0721] In one embodiment, the alkyne linker is given by formula (XVIII):

[0722] [ka] (In the formula, X is CH2, CH2(CH2) n (CH2CH2O) m CH2CH2, NHCO(CH2) n NHCO(CH2CH2O) m CH2CH2, OCH2(CH2) n and O(CH2CH2O) m (Selected from the group consisting of CH2CH2, n is selected from 1 to 10, and m is selected from 1 to 4) It is a compound of [the compound].

[0723] In one embodiment, the alkyne linker is a compound of formula (XVIII) (wherein X is CH2).

[0724] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is CH2(CH2) n The compound is (where 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 yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0725] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is (CH2CH2O) mThe compound is CH2CH2 (where m is selected from 1 to 4). In one embodiment, m is selected from 1 to 3. In another embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In yet another embodiment, m is 2. In yet another embodiment, m is 3. In yet another embodiment, m is 4.

[0726] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is NHCO(CH2) n The compound is (where 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 yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0727] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is NHCO(CH2CH2O) m The compound is CH2CH2 (where m is selected from 1 to 4). In one embodiment, m is selected from 1 to 3. In another embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In yet another embodiment, m is 2. In yet another embodiment, m is 3. In yet another embodiment, m is 4.

[0728] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is OCH2(CH2) nThe compound is (where 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 yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0729] In one embodiment, the alkyne linker is given by formula (XVIII) (wherein X is O(CH2CH2O) m The compound is CH2CH2 (where m is selected from 1 to 4). In one embodiment, m is selected from 1 to 3. In another embodiment, m is selected from 1 to 2. In a particular embodiment, m is 1. In yet another embodiment, m is 2. In yet another embodiment, m is 3. In yet another embodiment, m is 4.

[0730] In one embodiment, the alkyne linker is given by formula (XIX):

[0731] [ka] It is a compound of [the compound].

[0732] Therefore, in a preferred embodiment, the alkyne linker is a propargylamine.

[0733] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an azide group is of formula (XX):

[0734] [ka] (In the formula, X is (CH2) nCH2C=O and (CH2CH2O) m (Selected from the group consisting of CH2CH2=O, n is selected from 0 to 10, and m is selected from 0 to 4) It is a compound of [the compound].

[0735] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an azide group is of formula (XX) (wherein X is (CH2) n It is a compound of CH2C=O (where 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 particular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet another embodiment, n is 4. In yet another embodiment, n is 5. In yet another embodiment, n is 6. In yet another embodiment, n is 7. In yet another embodiment, n is 8. In yet another embodiment, n is 9. In yet another embodiment, n is 10.

[0736] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an azide group is of formula (XX) (wherein X is (CH2CH2O) m The compound is CH2CH2=O (where m is selected from 0 to 4). In some embodiments, m is selected from 0 to 3. In some embodiments, m is selected from 0 to 2. In some embodiments, m is selected from 0 to 1. In certain embodiments, m is 0. In certain embodiments, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In yet another embodiment, m is 4.

[0737] In one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an azide group is of formula (XXI):

[0738] [ka] It is a compound of [the compound].

[0739] Therefore, in one embodiment, the agent having an N-hydroxysuccinimide (NHS) moiety and an azide group is (2,5-dioxopyrrolidine-1-yl)2-azidoacetate.

[0740] In one embodiment, step a) includes reacting a sugar with a carbonate derivative, and then in an aprotic solvent, reacting the sugar activated by the carbonate derivative with an alkyne linker to produce an activated alkynyl sugar.

[0741] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.01 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0742] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.1 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0743] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.5 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0744] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0745] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 2 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0746] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 5 to 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0747] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.1 to 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0748] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of 0.5 to 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0749] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.01 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0750] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.1 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0751] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0752] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 0.5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0753] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 1 molar equivalent relative to the amount of sugar present in the reaction mixture.

[0754] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 2 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0755] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 5 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0756] In one embodiment, step a) includes reacting the sugar with a carbonate derivative in an amount of about 10 molar equivalents relative to the amount of sugar present in the reaction mixture.

[0757] In one embodiment, in step a), the isolated sugar is reacted with a carbonic acid derivative in an aprotic solvent.

[0758] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution containing dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution containing dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution containing dimethyl sulfoxide (DMSO).

[0759] In a preferred embodiment, the isolated sugar is reacted with a carbonate derivative in a solution containing dimethyl sulfoxide (DMSO).

[0760] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO).

[0761] In one embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethylacetamide. In another embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of N-methyl-2-pyrrolidone. In yet another embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of hexamethylphosphoramide (HMPA).

[0762] In a preferred embodiment, the isolated sugar is reacted with a carbonate derivative in a solution essentially consisting of dimethyl sulfoxide (DMSO).

[0763] In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethylformamide (DMF). In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethyl sulfoxide (DMSO).

[0764] In one embodiment, the isolated sugar is reacted with a carbonate derivative in dimethylacetamide. In another embodiment, the isolated sugar is reacted with a carbonate derivative in N-methyl-2-pyrrolidone. In yet another embodiment, the isolated sugar is reacted with a carbonate derivative in hexamethylphosphoramide (HMPA).

[0765] In a preferred embodiment, the isolated sugar is reacted with CDI in dimethyl sulfoxide (DMSO). In a certain embodiment, the isolated sugar is reacted with CDI in anhydrous DMSO.

[0766] Surprisingly, it was found that side reactions could be avoided by reacting the isolated sugar with CDI in an environment with a humidity level of approximately 0.1% to 1% (v / v). Therefore, in one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 1% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.5% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.4% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.3% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.2% (v / v) water.

[0767] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.8% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.4% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.2% to 0.3% (v / v) water.

[0768] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.8% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.3% to 0.4% (v / v) water.

[0769] Preferably, in one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing 0.1% to 0.5% (v / v) water.

[0770] In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.2% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.3% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.4% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.5% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.6% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.7% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.8% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in an aprotic solvent containing about 0.9% (v / v) water.

[0771] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.8% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.5% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.4% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.3% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.2% (v / v) water.

[0772] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.8% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.4% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.2% to 0.3% (v / v) water.

[0773] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.8% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.5% (v / v) water. In yet another embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.3% to 0.4% (v / v) water.

[0774] Preferably, in one embodiment, the isolated sugar is reacted with CDI in DMSO containing 0.1% to 0.5% (v / v) water.

[0775] In one embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.1% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.2% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.3% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.4% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.5% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.6% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.7% (v / v) water. In another embodiment, the isolated sugar is reacted with CDI in DMSO containing about 0.8% (v / v) water. In one embodiment, the isolated sugar is reacted with CDI in DMSO containing approximately 0.9% (v / v) water.

[0776] In one embodiment, the free carbon dioxide derivative is then quenched by adding water before the addition of the alkyne linker. The water can activate the free CDI.

[0777] Therefore, in one embodiment, water is added after the activation of the carbonic acid derivative. In one embodiment, water is added to bring the total water content of the mixture to about 1% to about 10% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 1% to about 5% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 1% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 2% (v / v). In one embodiment, water is added to bring the total water content of the mixture to about 5% (v / v).

[0778] After reacting sugar with a carbonate derivative, the carbonate derivative is finally quenched with water, and then the sugar, activated by the carbonate derivative, is reacted with an alkyne linker.

[0779] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of alkyne linker that is 0.01 to 10 molar equivalents (molar equivalents of RU) relative to the amount of polysaccharide repeating units of the activated sugar.

[0780] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of alkyne linker that is 0.1 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0781] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of alkyne linker that is 0.1 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0782] In one embodiment, step a) further includes reacting the sugar activated by the carbonate derivative with an amount of alkyne linker that is 0.5 to 2 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0783] In one embodiment, step a) further includes reacting a sugar activated by a carbonate derivative with an amount of alkyne linker that is 1 to 5 molar equivalents relative to the amount of polysaccharide repeating units of the activated sugar.

[0784] In the above embodiment, the carbonate derivative is CDI. In another embodiment, the carbonate derivative is CDT. In the above embodiment, the carbonate derivative is preferably DSC.

[0785] In one embodiment, the activation degree of the activated sugar after step a) is 0.5 to 50%. The activation degree of the alkynyl sugar is defined as the percentage of repeating units linked to the alkyne linker.

[0786] In one embodiment, the activation degree of the activated sugar after step a) is 1-30%. In another embodiment, the activation degree of the activated sugar after step a) is 2-25%. In yet another embodiment, the activation degree of the activated sugar after step a) is 3-20%.

[0787] In another embodiment, the activation degree of the activated sugar after step a) is 3-15%. In yet another embodiment, the activation degree of the activated sugar after step a) is 4-15%. In one embodiment, the activation degree of the activated sugar after step a) is 1-6%.

[0788] In one embodiment, the activation degree of the activated sugar after step a) is 3-6%. In another embodiment, the activation degree of the activated sugar after step a) is 10-15%. In a preferred embodiment, the activation degree of the activated sugar after step a) is 15-50%.

[0789] In one embodiment, the activation degree of the activated sugar after step a) is about 1%. In one embodiment, the activation degree of the activated sugar after step a) is about 2%. In one embodiment, the activation degree of the activated sugar after step a) is about 3%. In one embodiment, the activation degree of the activated sugar after step a) is about 4%. In one embodiment, the activation degree of the activated sugar after step a) is about 5%. In one embodiment, the activation degree of the activated sugar after step a) is about 6%. In one embodiment, the activation degree of the activated sugar after step a) is about 7%. In one embodiment, the activation degree of the activated sugar after step a) is about 8%. In one embodiment, the activation degree of the activated sugar after step a) is about 9%. In one embodiment, the activation degree of the activated sugar after step a) is about 10%. In one embodiment, the activation degree of the activated sugar after step a) is about 11%. In one embodiment, the activation degree of the activated sugar after step a) is about 12%. In another embodiment, the activation degree of the activated sugar after step a) is about 13%. In another embodiment, the activation degree of the activated sugar after step a) is about 14%. In another embodiment, the activation degree of the activated sugar after step a) is about 15%. In another embodiment, the activation degree of the activated sugar after step a) is about 16%. In another embodiment, the activation degree of the activated sugar after step a) is about 17%. In another embodiment, the activation degree of the activated sugar after step a) is about 18%. In another embodiment, the activation degree of the activated sugar after step a) is about 19%. In another embodiment, the activation degree of the activated sugar after step a) is about 20%.

[0790] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of 0.1 to 10 molar equivalents relative to the lysine on the carrier protein.

[0791] In one embodiment, step b) includes reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of 0.5 to 10 molar equivalents relative to the lysine on the carrier protein.

[0792] In one embodiment, step b) includes reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an azide group, in an amount of 1 to 5 molar equivalents relative to the lysine on the carrier protein.

[0793] In one embodiment, step b) includes reacting the carrier protein with an agent having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of 2 to 5 molar equivalents relative to the lysine on the carrier protein.

[0794] In one embodiment, step b) includes reacting the carrier protein with an amount of an agent having an N-hydroxysuccinimide (NHS) moiety and an azide group, in an amount of about 10 molar equivalents relative to the lysine on the carrier protein.

[0795] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 7.5 molar equivalents relative to the lysine on the carrier protein.

[0796] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 5 molar equivalents relative to the lysine on the carrier protein.

[0797] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 2 molar equivalents relative to the lysine on the carrier protein.

[0798] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 1 molar equivalent relative to the lysine on the carrier protein.

[0799] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 0.5 molar equivalents relative to the lysine on the carrier protein.

[0800] In one embodiment, step b) includes reacting the carrier protein with a drug having an N-hydroxysuccinimide (NHS) moiety and an azide group in an amount of about 0.1 molar equivalents relative to the lysine on the carrier protein.

[0801] In one embodiment, the activation degree of the activated carrier after step b) is 1 to 50. The activation degree of the activated carrier is defined as the number of lysine residues in the carrier protein that become linked to the drug having an N-hydroxysuccinimide (NHS) moiety and an azide group.

[0802] In one embodiment, the carrier protein is a CRM containing 39 lysine residues. 197 In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 30. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 5 to 20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 9 to 18. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is 8-11. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of the ) is 15-20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 5. In another embodiment, the activated carrier (CRM) after step b)197 The activation degree of ) is approximately 6. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 7. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 8. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 9. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 10. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 11. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 12. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 13. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 14. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 15. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 16. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 17. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 18. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 19. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 20. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 21. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 22. In another embodiment, the activated carrier (CRM) after step b) 197The activation degree of ) is approximately 23. In another embodiment, the activated carrier (CRM) after step b) 197 The activation degree of ) is approximately 24. In another embodiment, the activated carrier (CRM) after step b) 197 The activation level of ) is approximately 25.

[0803] In one embodiment, the carrier protein is an SCP or a fragment thereof. In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 50. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 5 to 50. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 7 to 45. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 5 to 15. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 20 to 30. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 30 to 50. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 30 to 40. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is 10 to 40. In a preferred embodiment, the activation degree of the activated carrier (SCP) after step b) is 15 to 25. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 5. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 7. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 10. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 13. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 15. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 20. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 26. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 30. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 35. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 37. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 40. In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 45.In another embodiment, the activation degree of the activated carrier (SCP) after step b) is approximately 50.

[0804] In one embodiment, the carrier protein is TT or a fragment thereof. In the above embodiment, the activation degree of the activated carrier after step b) may be 1 to 30. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 5 to 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 7 to 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is 10 to 20. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 5. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 7. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 10. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 12. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 15. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 20. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 25. In another embodiment, the activation degree of the activated carrier (TT) after step b) is about 30.

[0805] In one embodiment, the conjugation reaction c) is carried out in an aqueous buffer. In another embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst. In yet another embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as an oxidizing agent and a catalyst. In a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst and an ascorbate as an oxidizing agent. In one embodiment, THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine can be further added to protect the protein from side reactions. Therefore, in a preferred embodiment, the conjugation reaction c) is carried out in an aqueous buffer in the presence of copper(I) as a catalyst and an ascorbate as an oxidizing agent, and the reaction mixture further comprises THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) and aminoguanidine.

[0806] In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is 0.1 to 3. In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is 0.5 to 2. In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is 0.6 to 1.5. In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is 0.8 to 1. In a preferred embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is 0.8 to 1.2. In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is approximately 0.5. In one embodiment, the initial input ratio (weight / weight) of the activated alkynyl sugar to the activated azide-carrier in step c) is approximately 0.6. In one embodiment, the initial input rat...

Claims

1. The following iteration units: 【Chemistry 1】 (In the formula, n represents the number of repeating units; the O-acetyl group at position 3 of β-D-Galf is present in approximately 50% to 80% of the repeating units; the O-acetyl group at position 6 of β-D-Galf is present in approximately 20% to 40% of the repeating units; and the O-acetyl group at position 5 of β-D-Galf is present in approximately 1% to 10% of the repeating units.) A sugar conjugate containing isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar.

2. The sugar conjugate according to claim 1, wherein the O-acetyl group at position 3 of β-D-Galf is present in approximately 64% of the repeating units, the O-acetyl group at position 6 of β-D-Galf is present in approximately 30% of the repeating units, and the O-acetyl group at position 5 of β-D-Galf is present in approximately 6% of the repeating units.

3. A sugar conjugate containing isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeating units, the O-acetyl group at position 3 of β-D-Galf is present in approximately 25% to 60% of the repeating units, the O-acetyl group at position 6 of β-D-Galf is present in approximately 30% to 65% of the repeating units, and the O-acetyl group at position 5 of β-D-Galf is present in approximately 5% to 15% of the repeating units).

4. The sugar conjugate according to any one of claims 1 to 3, wherein all repeating units have one O-acetyl group on a β-D-Galf residue.

5. A sugar conjugate containing isolated Streptococcus pneumoniae (S. pneumoniae) serotype 21 sugar having repeat units of formula (VIII) (wherein n represents the number of repeat units, and the O-acetyl groups at the 3, 5, or 6 positions of β-D-Galf are present in approximately 0% to approximately 95% of the repeat units).

6. A serotype 21 sugar conjugate according to any one of claims 1 to 5, prepared by direct reductive amination.

7. A serotype 21 sugar conjugate according to any one of claims 1 to 5, prepared by click chemistry.

8. It contains a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and has the general formula (IV): 【Chemistry 2】 (where 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 is selected from the group consisting of, 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. The structure in square brackets represents the repeating unit of serotype 21-saccharide, where n represents the number of repeating units. A serotype 21 sugar conjugate possessing this characteristic.

9. It contains a serum-type 21-saccharide covalently conjugated to a carrier protein (CP) via a spacer, and has the general formula (IV) (wherein X is CH). ２ (CH ２ ) ｎ ' is , n' is 2, and X' is CH ２ O(CH ２ ) ｎ’’ CH ２ A serotype 21 sugar conjugate having C=O and n''=1.

10. It contains a serum type 21 sugar covalently conjugated to a carrier protein (CP) via a spacer, and has the general formula (V): 【Transformation 3】 (In the formula, the structure in square brackets represents the repeating unit of serotype 21-saccharide, and n represents the number of repeating units.) A serotype 21 sugar conjugate possessing this characteristic.

11. The sugar conjugate according to any one of claims 1 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae before conjugation is 100 kDa to 400 kDa.

12. The sugar conjugate according to any one of claims 1 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae before conjugation is 150 kDa to 400 kDa.

13. The sugar conjugate according to any one of claims 1 to 12, wherein the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 500 kDa to 15,000 kDa.

14. The sugar conjugate according to any one of claims 1 to 12, wherein the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 750 kDa to 7,500 kDa.

15. The sugar conjugate according to any one of claims 7 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 150 kDa to 400 kDa, and the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 800 kDa to 7,000 kDa.

16. The sugar conjugate according to claim 6, wherein the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2,000 kDa to 10,500 kDa.

17. The sugar conjugate according to claim 6, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 100 kDa to 400 kDa, and the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2000 kDa to 10,500 kDa.

18. The sugar conjugate according to any one of claims 1 to 17, wherein the degree of conjugation of the serotype 21 sugar conjugate is 2 to 15.

19. The sugar conjugate according to any one of claims 7 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae before conjugation is 150 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 800 kDa to 7,000 kDa, and the degree of conjugation of the serotype 21 sugar conjugate is 2 to 10.

20. The sugar conjugate according to claim 6, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 100 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2000 kDa to 10,500 kDa, and the degree of conjugation of the serotype 21 sugar conjugate is 2 to 10.

21. A sugar conjugate according to any one of claims 1 to 20, wherein the ratio (w / w) of serum type 21 sugars in the sugar conjugate to the carrier protein is 0.5 to 1.

5.

22. The sugar conjugate according to any one of claims 7 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 150 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 800 kDa to 7,000 kDa, and the ratio (w / w) of the serotype 21 sugar in the sugar conjugate to the carrier protein is 0.5 to 1.

1.

23. The sugar conjugate according to claim 6, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 100 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2000 kDa to 10,500 kDa, and the ratio (w / w) of the serotype 21 sugar in the sugar conjugate to the carrier protein is 0.5 to 1.

3.

24. The sugar conjugate according to any one of claims 1 to 23, wherein the serotype 21 sugar conjugate contains less than 25% of free serotype 21 sugars compared to the total amount of serotype 21 sugars.

25. The sugar conjugate according to any one of claims 7 to 10, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 150 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 800 kDa to 7,000 kDa, the ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate is 0.5 to 1.1, and the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar.

26. The sugar conjugate according to claim 6, wherein the weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 100 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2000 kDa to 10,500 kDa, the ratio (w / w) of serotype 21 sugar to carrier protein in the sugar conjugate is 0.5 to 1.3, and the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar.

27. The weight-average molecular weight (Mw) of the serotype 21-saccharide of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 150 kDa to 400 kDa, the serotype 21-saccharide conjugate has a weight-average molecular weight (Mw) of 800 kDa to 7,000 kDa, the ratio (w / w) of serotype 21-saccharide in the sugar conjugate to the carrier protein is 0.5 to 1.1, the serotype 21-saccharide conjugate contains less than 25% free serotype 21-saccharide compared to the total amount of serotype 21-saccharide, and 65% to 80% of the serotype 21-saccharide conjugate has a K value less than 0.3 or equal to 0.3 in the CL-4B column. ｄ A sugar conjugate according to any one of claims 7 to 10, having the following characteristics.

28. The weight-average molecular weight (Mw) of the serotype 21 sugar of Streptococcus pneumoniae (S. pneumoniae) before conjugation is 100 kDa to 400 kDa, the serotype 21 sugar conjugate has a weight-average molecular weight (Mw) of 2000 kDa to 10,500 kDa, the ratio (w / w) of serotype 21 sugar in the sugar conjugate to the carrier protein is 0.5 to 1.3, the serotype 21 sugar conjugate contains less than 25% free serotype 21 sugar compared to the total amount of serotype 21 sugar, and 65% to 80% of the serotype 21 sugar conjugate has a K content below or equal to 0.3 in the CL-4B column. ｄ A sugar conjugate according to claim 6, having the following characteristics.

29. The sugar conjugate according to any one of claims 1 to 28, wherein the carrier protein of the serotype 21 sugar sugar conjugate is selected from the group consisting of TT, DT, DT mutants, and C5a peptidase (SCP) derived from Streptococcus pneumoniae.

30. The sugar conjugate according to any one of claims 1 to 28, wherein the carrier protein of the serum type 21 sugar sugar conjugate is rizavidin [aa45-179J-GGGGSSS-SP1500-AAA-SP0785] (CP1).

31. The sugar conjugate according to any one of claims 1 to 28, wherein the carrier protein of the serotype 21 sugar sugar conjugate is Rhavi-linker-PdT(G294P)-linker-SP0435[aa62-185] fusion protein (SPP2).

32. The carrier protein of the serum type 21 sugar sugar conjugate is CRM １９７ The sugar conjugate according to any one of claims 1 to 28.

33. The sugar conjugate according to any one of claims 1 to 28, wherein the carrier protein of the serum type 21 sugar sugar conjugate is SCP.

34. An immunogenic composition comprising Streptococcus pneumoniae serotype 21 sugar according to any one of claims 1 to 33.

35. The immunogenic composition according to claim 34, further comprising a sugar conjugate derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F of Streptococcus pneumoniae.

36. The immunogenic composition according to claim 34, further comprising sugar conjugates derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, and 35B of Streptococcus pneumoniae.

37. Streptococcus pneumoniae sugars derived from serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23A, 23B, 23F, 24F, 33F, 35B, and 38 are CRM １９７ The immunogenic composition according to claim 34, wherein Streptococcus pneumoniae glycoserotype 3 is conjugated to SCP.

38. An immunogenic composition according to any one of claims 34 to 37, further comprising one adjuvant.

39. An immunogenic composition according to any one of claims 34 to 38 for use as a vaccine.

40. An immunogenic composition according to any one of claims 34 to 38 for use in a method for preventing infection by serotype 21 of Streptococcus pneumoniae in a subject, comprising the step of administering to the subject an immunologically effective amount of the immunogenic composition.

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