Saccharide-polypeptide conjugate compositions and methods of use thereof

Abstract

To provide saccharide-polypeptide conjugate compositions and to provide methods of use thereof.SOLUTION: Saccharide-polypeptide conjugates, compositions containing the same, methods of making and using the conjugates and compositions, and kits containing the same are disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to U.S. Provisional Patent Application No. 62 / 316,555, filed March 31, 2016; U.S. Provisional Patent Application No. 62 / 330,245, filed May 2, 2016; U.S. Provisional Patent Application No. 62 / 372,263, filed August 8, 2016; and U.S. Provisional Patent Application No. 62 / 373,807, filed August 11, 2016, each of which is incorporated herein by reference in its entirety. Summary of the Invention [Means for solving the problem]

[0002] A brief summary is provided herein to help understand the embodiments provided herein, but the embodiments are not limited to the content provided in this summary.

[0003] Provided herein is a pharmaceutical composition that can be present in unit dosage form and that comprises a plurality of at least eight unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof, conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). 1. A pharmaceutical composition, which can be present in unit dosage form, comprising a plurality of at least 29 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). 1. A pharmaceutical composition, which can be present in unit dosage form, comprising at least two immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof, conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype, one serotype being 6C and at least one further serotype being selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B).1. A pharmaceutical composition, which can be present in unit dosage form, comprising at least two immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof, conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype, one serotype being 20B and at least one further serotype being selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, and 38.

[0004] 1. A pharmaceutical composition comprising an immunogenic saccharide-polypeptide conjugate, which can be present in unit dosage form and individually comprises at least two unique capsular polysaccharides, fragments thereof, or combinations thereof conjugated to a polypeptide, wherein the at least two unique capsular polysaccharides, fragments thereof, or combinations thereof are derived from a serotype of Streptococcus pneumoniae selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may further comprise at least one unique immunogenic saccharide-polypeptide conjugate, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B).

[0005] Provided herein are methods comprising administering to a subject a first composition, wherein the first composition is any one of the pharmaceutical compositions described herein. Also provided herein are methods comprising administering to a subject a first composition and a second composition, wherein administering to the subject the first composition occurs at least about 4 weeks before or at least about 4 weeks after administering to the subject a second composition comprising an immunogenic saccharide-polypeptide conjugate comprising a second polypeptide, wherein the second polypeptide has at least about 70% length homology, at least about 70% sequence homology, or a combination thereof, to the polypeptide of the first composition.

[0006] Provided herein are methods comprising administering to a subject a second immunogenic saccharide-polypeptide conjugate composition at least four weeks after administration of a first immunogenic saccharide-polypeptide conjugate composition, wherein the second immunogenic saccharide-polypeptide conjugate composition comprises a first polypeptide having at least about 70% length homology, at least about 70% sequence homology, or a combination thereof, to a second polypeptide of the second immunogenic saccharide-polypeptide conjugate composition, wherein the first immunogenic saccharide-polypeptide conjugate composition comprises a first serotype and the second immunogenic saccharide-polypeptide conjugate composition comprises a second serotype, wherein the first serotype and the second serotype are different.

[0007] Further provided herein is a method of making a composition, the method comprising contacting an immunogenic saccharide-polypeptide conjugate of any one of the described pharmaceutical compositions with an excipient, an adjuvant, or any combination thereof.

[0008] Provided herein are kits that include any of the pharmaceutical compositions described herein or any of the pharmaceutical compositions contained in a container described herein.

[0009] Provided herein are methods of making the compositions, methods of immunizing with the compositions, and methods of vaccinating with the compositions. Incorporation by Reference

[0010] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0011] The novel features of the invention are set forth with particularity in the appended claims. A more complete understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a flow diagram for the general process for the manufacture of saccharide-peptide conjugate vaccines.

[0013] [Figure 2] FIG. 2 shows a flow diagram for the process for cell banking of bacterial strains.

[0014] [Figure 3] FIG. 3 shows a flow diagram for the process for producing, isolating, and purifying CRM197.

[0015] [Figure 4] FIG. 4 shows a flow diagram for a fermentation process for isolating polysaccharides from a strain of Streptococcus pneumoniae.

[0016] [Figure 5] FIG. 5 shows a flow diagram for the conjugation process for conjugating polysaccharide and CRM197.

[0017] [Figure 6] FIG. 6 shows a flow diagram for a process for formulating a polysaccharide-polypeptide conjugate vaccine.

[0018] [Figure 7] Figure 7 shows the projected expanded coverage of Streptococcus pneumoniae polypeptide conjugate vaccines containing Streptococcus pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B (ePCV; LiffeyVax serotypes) against invasive pneumococcal disease (IPD) in the United States and Canada when administered in a vaccination regimen with Prevnar 13 (PCV13). Prevnar 13 alone provides only approximately 46.1% protection against IPD compared to an overall coverage of 90% when administered in a vaccination regimen with ePCV.

[0019] [Figure 8]Figure 8 shows the projected expanded coverage of invasive pneumococcal disease (IPD) in the United States and Canada provided by a Streptococcus pneumoniae polypeptide conjugate vaccine containing Streptococcus pneumoniae serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 35B (Liffey / Vax ePCV8) when administered in a vaccination regimen with Prevnar 13 (PCV13) or a vaccine containing Prevnar 13 serotypes and seven additional serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) (PCV20). Prevnar 13 alone protects against IPD in only approximately 46.1%, while the addition of the seven serotypes of PCV20 is predicted to provide approximately 21% more IPD protection. The addition of eight additional serotypes in Liffey / Vax ePCV8 is predicted to confer approximately 23% more protection against IPD when administered in a vaccination regimen with PCV20.

[0020] [Figure 9]Figure 9 shows the projected expanded coverage of invasive pneumococcal disease (IPD) in the United States and Canada provided by a Streptococcus pneumoniae polypeptide conjugate vaccine containing Streptococcus pneumoniae serotypes 6C, 9N, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 34, 35F, 35B, and 38 (ePCV8+7) when administered in a vaccination regimen with Prevnar 13 (PCV13) or a vaccine containing Prevnar 13 serotypes and seven additional serotypes (8, 10A, 11A, 12F, 15B, 22F, and 33F) (PCV20). While Prevnar 13 alone provides protection against only approximately 46.1% of IPD, the addition of the seven serotypes of PCV20 is predicted to provide approximately 21% more IPD protection. The addition of seven additional serotypes in Liffey / Vax ePCV8 (Liffey / Vax ePCV8+7) (see Figure 8 for ePCV8) is predicted to provide approximately 29% more IPD protection when administered in a vaccination regimen with PCV20.

[0021] [Figure 10-1] Figures 10A, 10B, 10C, 10D, 10E, and 10F show the annotated capsular polysaccharide (cps) loci for various serotypes of S. pneumoniae (Bentley et al., Genetic Analysis of the Capsular Biosynthetic Locus from all 90 Pneumococcal Serotypes. PloS Genet 2(3):31 (2006)). [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description Some aspects are described below with reference to the application of examples for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the requirements described herein. However, those skilled in the art will readily recognize that the requirements described herein can be implemented without one or more of the specific details or using other methods. The requirements described herein are not limited to the illustrated order of acts or events, as some acts may occur in a different order and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to execute the methodology in accordance with the requirements described herein.

[0023] The terms used herein are for descriptive purposes only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in the same manner as "comprising."

[0024] The terms "about" or "approximately" can mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, depending in part on how a particular value is measured or determined, i.e., the limitations of the measurement system. For example, relative to practice in the art, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range encompassing plus or minus up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values ​​are described in this application and in the claims, unless otherwise stated, the term "about" can mean within an acceptable range of error for the particular value.

[0025] When used in reference to pharmaceutical compositions, "unit dose" can refer to a unit suitable as a unitary dosage for humans, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect. In some cases, a unit dose can contain a diluent, i.e., a carrier, or vehicle. In some cases, a unit dose can be physically separate.

[0026] The term "saccharide" can refer to a saccharide, oligosaccharide, or polysaccharide. For example, a saccharide can refer to a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, octasaccharide, nonasaccharide, or decasaccharide. An oligosaccharide can refer to a disaccharide to decasaccharide. A polysaccharide can refer to a sugar greater than a decasaccharide.

[0027] The term "polypeptide" can refer to a single linear chain of amino acids held together by amide bonds. A polypeptide can refer to at least two amino acids. A polypeptide can be an oligopeptide, and the term "oligopeptide" can refer to a short chain of 2 to 20 amino acids joined by amide bonds. A polypeptide can be a protein, and the term "protein" can refer to a short chain of 50 or more amino acids held together by amide bonds.

[0028] The term "immune response" can refer to the production of cytokines and / or antibodies with a certain degree of specificity to an antigen after administering the antigen to a subject. In some examples, the term "immune response" can refer to changes in immune cells or changes caused by immune cells after administering the antigen to a subject. The immune response can be T cell-independent, T cell-dependent, or both.

[0029] The term "homology" can refer to the percent sequence identity between a particular nucleic acid or amino acid sequence and another nucleic acid or amino acid sequence. First, a nucleic acid or amino acid sequence can be compared to another sequence using the BLAST2 sequence (Bl2seq) program from the standalone version of BLASTZ, which contains BLASTN version 2.0.14 and BLASTP version 2.0.14. BLASTN can be used to compare nucleic acid sequences, and BLASTP can be used to compare amino acid sequences. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. The percent sequence identity or homology can be determined by dividing the number of matches by the sequence length represented in the identified sequence or by the concatenated length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence represented in the identified sequence), and then multiplying the resulting value by 100%.

[0030] The term "length homology" can refer to the percent length identity between the length of a particular polypeptide and the length of another polypeptide. "Length homology" can, in some instances, be calculated by dividing the number of amino acids in a first peptide chain by the number of amino acids in a second peptide chain and multiplying the result by 100%. The amino acids in the chains can be those that form the peptide backbone.

[0031] With respect to polypeptides, the term "length" can refer to a count of the number of amino acids along an amino acid chain.

[0032] The term "immunogenic fragment" refers to a fragment that, when conjugated to a polypeptide and administered to a subject, elicits an immune response. In some instances, the fragment may be a fragment of a capsular polysaccharide. In other instances, the polypeptide may be a fragment of a CRM. 197 It is possible.

[0033] Immunogenicity may be required to be placed in front of any term in this specification, but it is not necessary, and may mean that when administered to a subject, which may be human or animal, for example, dog, cat, rat, mouse, sheep or monkey, it can cause an immune response in the animal.For example, dog can be Canis lupus familiarius, cat can be Felis catus, rat can be Rattus norvegicus, mouse can be Mus musculus, sheep can be Ovis aries, or monkey can be Simia inuus.Administration can be, for example, subcutaneous, intramuscular, or rectal, for example, in the form of suppository.

[0034] In some embodiments, a unique immunogenic saccharide-polypeptide conjugate may differ in at least one aspect from other unique immunogenic saccharide-polypeptide conjugates, e.g., among a plurality of other unique immunogenic saccharide-polypeptide conjugates. For example, a unique immunogenic saccharide-polypeptide conjugate may differ by the saccharide or fragment thereof it contains, or by the polypeptide it contains, or both, in that it may be considered a unique immunogenic saccharide-polypeptide conjugate. In some instances, a unique immunogenic saccharide-polypeptide conjugate to be unique does not simply need to be 100% identical.

[0035] With respect to the toxic activity of a polypeptide, the term "partially ameliorate" may refer to a reduction in the toxicity of the polypeptide compared to the wild-type version of the polypeptide.

[0036] The term "molecular weight" can be number average molecular weight or weight average molecular weight.

[0037] The term "plurality" may mean two or more.

[0038] The term "subject" may refer to a human or an animal. A subject may be a subject in need thereof.

[0039] Bacteria can incorporate saccharides into their surface structures. For example, Streptococcus pneumoniae (S. pneumoniae) can contain saccharides in its surface structure. The saccharide can be an antigen that can induce an immune response in a host, such as an animal or a human. For example, B cells can produce antibodies against the saccharide. This response can be a T cell-independent immune response. The immune response can be induced by full-length native polysaccharides, oligosaccharides, or fragments thereof. The immune response can be induced by full-length native capsular polysaccharides or fragments thereof. Other bacteria that can contain saccharides are Neisseria meningitidis (N. meningitidis), Salmonella enterica (S. enterica), and Haemophilus influenzae (H. influenzae).

[0040] Saccharides present on bacteria can be used to classify bacteria into subclasses or serotypes. More specifically, a serotype can refer to the type of microorganism determined by its constituent antigens. The constituent antigens can be various saccharides, such as various capsular polysaccharides or immunogenic fragments thereof. Some non-limiting examples of S. pneumoniae include, but are not limited to, serogroups 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 22F, 23F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B).

[0041] Conjugating a saccharide to a polypeptide can enhance the immune response to the saccharide by inducing a T cell-dependent response. Saccharides alone may not stimulate T cells because, for example, the saccharide is not loaded onto the major histocompatibility complex (MHC) of antigen-presenting cells. However, peptides can be loaded onto MHC. Thus, by conjugating a saccharide to a polypeptide, the saccharide-polypeptide conjugate can then be loaded onto MHC and recognized by T cells, stimulating a T cell response. The T cells can then stimulate a more vigorous immune response and promote more rapid and long-lasting immunological memory. Provided herein are saccharide-polypeptide conjugates, which can be used to induce an immune response and may also be referred to as immunogenic saccharide-polypeptide conjugates. In some instances, the immunogenic saccharide-polypeptide conjugates can provide immune protection against the saccharide or organisms containing the saccharide. In other instances, the immunogenic saccharide-polypeptide conjugates can be part of a vaccine. Compositions of saccharide-polypeptide conjugates

[0042] The immunogenic saccharide-polypeptide conjugate composition can be a saccharide conjugated to a polypeptide. The immunogenic saccharide-polypeptide conjugate can be a capsular polysaccharide conjugated to a polypeptide. The immunogenic saccharide-polypeptide conjugate composition can be a capsular polysaccharide or a fragment of a capsular polysaccharide conjugated to a polypeptide. The immunogenic saccharide-polypeptide conjugate can comprise a saccharide antigen coupled to one or more polypeptides. The immunogenic saccharide-polypeptide conjugate can comprise one unique saccharide antigen coupled to a polypeptide. The immunogenic saccharide-polypeptide conjugate can comprise two or more unique saccharide antigens coupled to a polypeptide. The saccharide antigens can elicit an immune response.

[0043] The saccharides can be from any bacteria and fungi, and any bacteria and fungi can incorporate saccharides into their surface structure. For example, the saccharides can be from S. pneumoniae. Further examples include saccharides from N. meningitidis, S. enterica, or H. influenzae. The saccharides can be full-length, native polysaccharides, oligosaccharides, or fragments thereof. The saccharides can be in any form that induces an immune response. The saccharides can be capsular polysaccharides or fragments thereof. The saccharides can be derived from subclasses or serotypes. Some non-limiting examples of saccharides from S. pneumoniae serotypes include, but are not limited to, 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 22F, 23F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20. Serogroup 20 can include serotypes 20A and 20B. S. pneumoniae capable of producing serotype 20A polysaccharide can be identified by a mutated whaF allele compared to an intact whaF allele of S. pneumoniae capable of producing serotype 20B polysaccharide. Table 1 shows exemplary repeating unit polysaccharide structures for various S. pneumoniae serotypes. Figures 11A, 11B, 11C, 11D, 11E, and 11F show annotated capsular polysaccharide (cps) loci for various S. pneumoniae serotypes (Bentley et al., Genetic Analysis of the Capsular Biosynthetic Locus from all 90 Pneumococcal Serotypes. PloS Genet 2(3):31 (2006)). [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

[0044] Saccharides can be isolated from bacteria. Saccharides can be isolated from bacteria and then conjugated to polypeptides to form saccharide-polypeptide conjugates. Any method can be used to isolate unconjugated polysaccharides. For example, polysaccharide serotypes can be grown in soy-based media. Individual polysaccharides can then be purified by steps including centrifugation, precipitation, and ultrafiltration. See U.S. Patent Application Publication No. 2008 / 028638 and U.S. Patent No. 5,847,112. Other methods for purifying polysaccharides are described in U.S. Patent Nos. 4,686,102; 4,242,501; 4,221,906; 5,623,057; and 5,847,112. Alternatively or additionally, polysaccharides can be isolated from two or more serotypes simultaneously, which can produce a mixture of polysaccharide serotypes with the desired polysaccharide already combined. Furthermore, these isolated polysaccharides can be sorted by size by microfluidization. Polysaccharides can be sorted by size to reduce the viscosity of the polysaccharide sample and / or improve filterability for the conjugate sample. The saccharide-polypeptide conjugate can include a saccharide or fragment thereof at least partially embedded in one or more polypeptides. The one or more polypeptides can have one or more crosslinks. The at least partially embedded saccharide or fragment thereof can be, but need not be, covalently bound to the at least one polypeptide when at least partially embedded in the at least one polypeptide.

[0045] Any of the S. pneumoniae strains for isolating and purifying polysaccharides as described herein may be provided by either the CDC (USA) or CCUG (Gothenburg). Alternatively, any of the polysaccharides described herein may be isolated and purified from any source (e.g., any laboratory) where serotyping and other genetic sequencing are used to identify the specific strain from which the polysaccharide is isolated and purified. A vial of S. pneumoniae strain is inoculated and then allowed to grow overnight on an agar plate. A single colony can be selected from this plate and streaked onto a new agar plate for overnight growth. This process can be repeated three times to purify the strain before inoculating and incubating the flask. After the inoculated flask has been grown for a sufficient time, the contents of the flask can be harvested by centrifugation. The product of the centrifugation can be resuspended in approximately 20% glycerol medium and then aliquoted into cryogenic vials. The cryogenic vials contain cell banks and can be stored at -70°C until use. A strain from the cell bank can then be used to isolate and purify the polysaccharide of that strain using a fermentation process.

[0046] Polysaccharides from S. pneumoniae can be isolated and purified from cell bank strains. As described above, strains are provided by either the CDC (USA) or CCUG (Gothenburg) and can then be used to generate a cell bank, as described above. Alternatively, strains can be provided by any source (e.g., any laboratory) where serotyping or other genetic sequencing is used to identify the serotype of the strain. Cell bank vials are generated as described above and used to inoculate flasks and grow overnight. This culture is used to inoculate new cultures for fermentation in a fermentor. This culture is grown under conditions appropriate for the strain. After fermentation, the cells are inactivated and then treated with deoxycholate to rupture the cells and release the capsular polysaccharide. The cell-free broth is then harvested by centrifugation to remove debris and a depth filtration step through a 0.45 μm filter. The product is then purified, and the polysaccharide size is determined. Quality control checks are performed on the product, including purity assays and confirmation of strain inactivation (no growth after plating). Any of the polysaccharides described herein can be isolated and purified using this method.

[0047] The saccharides or polysaccharides of the present invention are depolymerized (sized) to a final range of 100-500 kDa.

[0048] Saccharides that can be used in saccharide-polypeptide conjugates can also be obtained from commercial sources. For example, individual polysaccharide serotypes are available in powder form. These polysaccharides can be dissolved in water and incubated with salt to dissociate residual impurities, which can then be removed by a filtration step. The purified polysaccharide serotypes can then be conjugated to polypeptides.

[0049] The polypeptide of the immunogenic saccharide-polypeptide conjugate can be an oligopeptide. The polypeptide can be a carrier protein. The polypeptide can be any polypeptide that allows for conjugation or coupling of a saccharide and that will exhibit the conjugate or coupling so as to induce a protective immune response against the saccharide. The polypeptide can be any polypeptide that allows for conjugation or coupling of a capsular polysaccharide or a fragment of a capsular polysaccharide and that will exhibit the conjugate or coupling so as to induce a protective immune response against the capsular polysaccharide or a fragment of a capsular polysaccharide. The polypeptide can be a CRM 197 , tetanus toxoid, diphtheria toxoid, cholera toxoid, pertussis toxoid, inactivated or mutated pneumococcal pneumolysin, pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, PspA, or any fragment thereof. The polypeptide can be one in which the toxic activity of the polypeptide can be at least partially mitigated. Examples of polypeptides suitable for use as saccharide-polypeptide conjugates are described in U.S. Patent No. 8,808,707 and include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids with other amino acids, such as serine, threonine, or tyrosine, that impart desired solubility characteristics to the resulting loaded carrier and conjugate. [ka] CRM with 100% sequence identity 197The polypeptide may be a CRM having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO:1. 197 The polypeptide may be a modified CRM as disclosed in U.S. Patent Application Publication No. 14 / 329,758. 197 The polypeptide can be a CRM having unnatural amino acid substitutions as disclosed in U.S. Patent Application Publication No. 14 / 328,532. 197 It is possible.

[0050] Additionally, suitable polypeptide carriers can include bacterial toxins, toxoids, or inactivated toxins. Toxoids can be bacterial toxins that have been made less toxic or inactivated while retaining other properties, typically immunogenicity. As a class, bacterial toxins and their derivatives can be highly immunogenic. Polypeptides derived from bacterial toxins can be effective in eliciting an immune response to the saccharide of the conjugate. Steps can be taken (e.g., by chemical and / or genetic means) to make the toxin non-toxic and safe for administration to mammals. Currently, examples of such bacterial toxin-derived polypeptides that may be commonly used in vaccine compositions and that can be used in the immunogenic saccharide-polypeptide conjugates described herein include diphtheria and tetanus toxoids and their variants (e.g., DT, CRM). 197 , TT), cholera toxoid, pertussis toxoid, inactivated or mutated pneumococcal pneumolysin, pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococci, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M. catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), H. Examples of polypeptide fragments include protein D from H. influenzae, PspA, or fragments of either. Polypeptide fragments can be used to elicit a protective immune response against the capsular polysaccharide or fragments of the capsular polysaccharide of the immunogenic saccharide-polypeptide conjugate. These fragments can include T-helper epitopes.

[0051] CRM 197 teeth, [ka] It is a non-toxic variant of diphtheria toxin with a single amino acid substitution of glutamic acid for glycine at position 52 of the amino acid sequence of CRM. 197 The CRM may be 58-59 kDa in size. 197 The CRM may be 58.4 kDa in size. 197 CRM can be produced, isolated and purified from cell banks. 197 Vials from the cell bank are used to inoculate new flasks and ferment. The fermented cultures can be centrifuged, the contents harvested, clarified, and filtered using a 0.2 μm filter. The filtrate can then be concentrated and diafiltered. 197 The CRM can then be purified further by filtration using microtubes or membranes of appropriate pore size. 197 This can be used for the isolation and purification of CRM, which can be subsequently precipitated with ammonium sulfate and filtered again using a 0.45 μm filter. 197 can be further isolated and purified from this product by hydrophobic interaction chromatography, followed by CRM 197 The isolated and purified CRM can be concentrated and diafiltered into the desired buffer. The final step can be filtration through a 0.2 μm filter. 197 The isolated and purified CRM can be stored at -70°C or below until use in combination with a saccharide or polysaccharide. 197 Quality control inspections may be carried out on the

[0052] The polypeptide can be conjugated to an immunogenic serotype 1 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to an immunogenic serotype 2 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to an immunogenic serotype 3 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to an immunogenic serotype 4 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to an immunogenic serotype 5 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to an immunogenic serotype 6A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 6B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 6C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 7F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 8 isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 9N isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 9V isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the immunogenic serotype 10A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof.The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 11A isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 12F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 14 isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 15A isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 15B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 15C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 16F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 17F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 18C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 19A isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 19F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 20A isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the immunogenic serotype 20B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof.The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 22F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 23A isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 23B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 23F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 24F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 24B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 31 isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 33F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 34 isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 35F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 35B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. The polypeptide can be conjugated to the capsular polysaccharide of immunogenic serotype 38 isolated from S. pneumoniae, a fragment thereof, or a combination thereof.

[0053] In some embodiments, the CRM 197 The CRM can be conjugated to an immunogenic serotype 1 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof.197 The CRM can be conjugated to an immunogenic serotype 2 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to an immunogenic serotype 3 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 4 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 5 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 6A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 6B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 6C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 7F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 8 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 9N isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 9V isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 10A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197The CRM can be conjugated to the immunogenic serotype 11A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 12F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 14 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 15A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 15B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 15C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 16F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 17F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 18C isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 19A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 19F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 20A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197The CRM can be conjugated to the immunogenic serotype 20B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 22F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 23A capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 23B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 23F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 24F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 24B capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 31 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 33F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the immunogenic serotype 34 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 35F isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197 The CRM can be conjugated to the capsular polysaccharide of immunogenic serotype 35B isolated from S. pneumoniae, a fragment thereof, or a combination thereof. 197can be conjugated to the immunogenic serotype 38 capsular polysaccharide isolated from S. pneumoniae, a fragment thereof, or a combination thereof.

[0054] In further embodiments, any of the saccharides described herein can be conjugated to any of the polypeptides described herein. Any of the immunogenic saccharide-polypeptide conjugates described herein can be isolated and purified. Methods for conjugating saccharides and polypeptides to form saccharide-polypeptide conjugates

[0055] The saccharide can be conjugated to the polypeptide, for example, by chemical or biological synthesis. The saccharide can be a polysaccharide, a capsular polysaccharide, or an immunogenic fragment thereof. The capsular polysaccharide or a fragment of a capsular polysaccharide can be conjugated to the polypeptide by chemical or biological synthesis.

[0056] For example, capsular polysaccharides or immunogenic fragments thereof can be chemically activated and then reacted with a polypeptide. For example, capsular polysaccharides or fragments of capsular polysaccharides can be reacted with either ammonia or diaminoethane to generate free terminal amino groups. The amino groups can then be reacted with excess disuccinimidyl ester of adipic acid to introduce activated succinimidyl ester groups, which can then be reacted with free amino groups on a polypeptide to form covalent amide bonds.

[0057] Polysaccharides and polypeptides can be conjugated using 1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP) chemistry. Using CDAP, a wide range of acidic, neutral, and other classes of polysaccharides can be activated and then derivatized with diamines, dihydrazides, or other functional groups in preparation for coupling to proteins. In CDAP, the cyanylating reagent 1-cyano-dimethylaminopyridinium tetrafluoroborate can be used to synthesize polysaccharide-protein conjugates. The cyanylation reaction can be performed under relatively mild conditions, which avoids hydrolysis of alkaline-sensitive polysaccharides. This synthesis may allow for direct coupling to supports. Polysaccharides can be dissolved in water or saline. CDAP can be dissolved in acetonitrile and immediately added to the polysaccharide solution. CDAP can react with the hydroxyl groups of polysaccharides to form cyanate esters. These cyanate esters are highly reactive and can result in activated polysaccharides. This can be done at pH 9-10. After this activation step, the polypeptide can be added. The amino groups of lysines on polypeptides can react with activated polysaccharides to form covalent isourea linkages. This conjugation step can occur under mildly alkaline conditions, such as pH 7-9. In some cases, the epsilon amine of lysines exhibits a strong pH dependency, allowing the formation of stable O-alkyl-isourea linkages. After the coupling reaction, excess amino-containing reagents, such as glycine, can be added to quench any remaining activated functional groups. CDAP activation requires only a few minutes, and conjugation can be completed in several hours. The conjugate can then be passed through a gel permeation column to remove unreacted polypeptide and residual reagents.

[0058] In some embodiments, CDAP is made up to 100 mg / ml in acetonitrile and can be stored at -20°C for up to one month. CDAP is slowly pipetted into a vortexed solution of polysaccharide in water (rapid addition of the organic cosolvent precipitates the polysaccharide), and after 20 seconds, a volume of 0.2 M aqueous triethylamine (TEA) solution equal to the volume of CDAP used can be added. At 2.5 minutes, a molar excess of 0.5 M hexanediamine in either 0.1 M sodium borate (pH 9.3) or 0.75 M HEPES (pH 7.5) can be added. The reaction mixture can be allowed to stand overnight at 4°C, desalted on either a P-6DG or P-6 cartridge, equilibrated with saline, and then further dialyzed against saline. The extent of derivatization with hexanediamine can be determined using the TNBS assay for primary amines; absorbance is measured at 11,000 M. -1 The polysaccharide concentration can be determined as described by Monsigny et al. using the corresponding polysaccharide as a standard.

[0059] In some embodiments, the polysaccharide can be dissolved in 10 mg / ml saline. At time t=0, CDAP (100 mg / ml in acetonitrile) can be added (1 mg CDAP per mg polysaccharide) while vortexing. After 30 seconds, 0.2 M TEA can be added (10 μl per mg polysaccharide) to raise the pH. After 2.5 minutes, the pH can be readjusted to the coupling pH, and an amine or hydrazide reagent or polypeptide can be added in an appropriate buffer. After an overnight reaction, the solution can be dialyzed and / or desalted on a P6DG column. The polysaccharide-polypeptide conjugate can be fractionated on an S400HR column, which can be equilibrated with either saline or PBS. HPLC can be performed on an HPLC size-exclusion column using a Waters 626 pump, monitored at 280 nm, and equipped with a refractive index detector. Samples can be filtered before chromatography using a Millex Ultrafree MC device.

[0060] In some embodiments, for coupling at low pH, the amines of a polypeptide can be functionalized with hydrazides using the following two-step method: 5 mg of a 12.5 mg / ml polypeptide in 0.19 M HEPES, 2.5 mM EDTA, pH 7.3 can be iodoacetylated by adding a 15-fold molar excess of SIA relative to the molar amount of polypeptide. After 2 hours of reaction in the dark, the solution can be desalted on a 1 x 15 cm P6DG column, equilibrated with 2 mM MES, 0.2 M NaCl, pH 5.9, and the void volume fraction can be concentrated on a Centricon 50 device. 30 μl of 0.1 M DTPH in water can be reduced to 3-mercaptopropionylhydrazide by adding 25 μl of 0.1 M TCEP in water, which can then be added to the iodoacetylated polypeptide, and the pH can be adjusted to approximately 5. After 2 hours of reaction, the reagents can be removed by desalting again and concentrating to 10 mg / ml using a Centricon 50 device. TNBS can be used to determine the number of hydrazides per mole of polypeptide. To couple the hydrazide-derivatized polypeptide, 3 mg of polysaccharide at 10 mg / ml in saline can be activated by adding 15 μl of 100 mg / ml CDAP in acetonitrile, followed 30 seconds later by 30 μl of 0.2 M TEA. After 2.5 minutes, the pH can be lowered to 5.5 by adding 100 μl of 1 M MES (pH 5.5), and the activated polysaccharide can be added to 3.8 mg of polypeptide-hydrazide. After overnight reaction at 4°C, the reaction can be quenched by adding 50 μl of 1 M ethanolamine. This solution can be applied to an S400HR column, which can be equilibrated with PBS, and the void volume fractions can be pooled. Sterile filtration can be performed through a Millex GB 0.2 μm filter.

[0061] In some embodiments, CDAP is used to separate polysaccharides and CRMs. 197can be conjugated. Polysaccharide (10 mg) can be dissolved in 1.0 mL of 0.1 M sodium borate buffer (pH 9.0) and activated by adding 50 μL of CDAP solution in acetone:water (6:4). The pH can be immediately adjusted and maintained at 9.0 by continuously adding 0.1 M NaOH solution dropwise. The mixture can be stirred for 5 minutes, then 1.4 mL of 0.05 M HCl can be added, stirred briefly, and then allowed to stand for 1.5 minutes. This mixture can be quickly passed through a PD-10 column equilibrated in 0.1 M sodium bicarbonate buffer (pH 8.3). An aliquot can be used to determine the formation of cyanate esters, and the remainder of the preparation can be used immediately for coupling purposes. Polypeptide (15 mg) can be dissolved in 1.0 mL of 0.1 M NaHCO3 buffer (pH 8.3) and added to the activated polysaccharide solution. This mixture can be gently stirred at room temperature for 8 hours. A 1.0 mL volume of 0.1 M Tris-HCl buffer (pH 8.6) was added to block any remaining activation sites on the unreacted polysaccharide, followed by an additional 1 hour of incubation. The final solution can be lyophilized and stored until use in the purification procedure. The progress of the conjugation can be analyzed by high-performance size-exclusion chromatography (HPSEC) using a Zorbax GF-250 column and monitored at 280 nm and 210 nm. For example, aliquots of the reaction mixture can be obtained every 10 minutes and every hour for up to 4 hours. The column can be equilibrated in 0.025 M phosphate buffer (pH 7.0) supplemented with 0.13 M NaCl and calibrated with the following proteins: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), and ribonuclease (13.7 kDa). The void volume can be determined using Blue Dextran. Samples (10 μl aliquots) can be injected onto the column at a flow rate of 0.5 mL / min.The column can be washed with equilibration buffer before and after analysis. The conjugation reaction progress (%) can be measured by comparing the conjugate peak area to the free protein peak area according to the following equation: (conjugate peak area / (conjugate peak area+free protein peak area))×100, using detection at a wavelength of 280 nm.

[0062] In some embodiments, the polysaccharide can be activated by CDAP chemistry in a CDAP solution. The polysaccharide can be mixed with the CDAP solution and then incubated at an appropriate temperature for polysaccharide activation. 197 The activated polysaccharide can then be obtained by adding a calculated amount of glycine or any other suitable quenching reagent. The conjugation reaction mixture can then be filtered, diafiltered, and concentrated to obtain the polysaccharide-CRM. 197 The polysaccharide-CRM conjugate can then be prepared. 197 The conjugate can be purified by gel filtration chromatography. 197 Fractions containing the conjugate can be pooled, concentrated, and then filtered through a 0.2 μm filter. The product can be stored at 2-8 °C.

[0063] As another example, capsular polysaccharides or immunogenic fragments thereof can be activated by periodate oxidation. Sodium periodate converts diols (two adjacent carbons with hydroxyl groups) into aldehydes (C=O), breaking the C-C bond in the process. Therefore, depending on the structure, periodate activation can fragment capsular polysaccharides or immunogenic fragments thereof, opening the sugar ring structure. If a diol is present within the ring, the sugar ring is opened, possibly altering the conformation of the polysaccharide. If a diol is present in a glycerol or ribitol side chain, the side chain can be lost. The reactive aldehyde group can then condense with free amino groups on polypeptides to form stable secondary amines in the presence of sodium cyanoborohydride. Condensation of the aldehyde with the epsilon-amino group of lysine is a slow process that can take several days. The remaining aldehyde groups can then be quenched using sodium borohydride, converting them back to hydroxyls.

[0064] Another example of coupling polypeptides to polysaccharides is based on the thioether linkage of 2-iminothiolane (2-IT) or cystamine-activated polysaccharides to bromoacetylated polypeptides such as tetanus toxoid. [ka]

[0065] Scheme I above depicts the end point of coupling of a polysaccharide (PS) to a protein (PROT) via a thioether bond. (A) Amination via the reducing end of the polysaccharide using ammonium chloride in the presence of cyanoborohydride ion. (B) Thiolation of the aminated polysaccharide (I) with 2-iminothiolane (2-IT). (C) Alternative thiolation of the polysaccharide via reductive amination with cystamine with simultaneous cleavage of disulfide bonds. (D) Bromoacetylation of the protein's amino groups using N-hydroxysuccimidyl bromoacetate. (E) Coupling of a polysaccharide derivatized with 2-IT (II) to a bromoacetylated protein (IV). (F) Alternative coupling of a polysaccharide derivatized with cystamine (III) to a bromoacetylated protein (IV).

[0066] As another example, immunogenic saccharide-polypeptide conjugates can be produced using bacteria such as Escherichia coli (E. coli). The immunogenic saccharide-polypeptide can comprise a capsular polysaccharide at least partially embedded in the polypeptide. The immunogenic saccharide-polypeptide can comprise a capsular polysaccharide chemically cross-linked to the polypeptide. The immunogenic saccharide-polypeptide can comprise a capsular polysaccharide at least partially chemically cross-linked to the polypeptide. Capsular polysaccharides of serotypes from S. pneumoniae or fragments of capsular polysaccharides can be conjugated to the polypeptide by chemical or biological synthesis. One saccharide can be conjugated to one polypeptide. Two or more saccharides can be conjugated to one polypeptide. The saccharides can be of the same serotype. The saccharides can be of different serotypes. One capsular polysaccharide or an immunogenic fragment thereof can be conjugated to one polypeptide. Two or more capsular polysaccharides or fragments thereof can be conjugated to one polypeptide. The capsular polysaccharides or fragments thereof can be of the same serotype. The capsular polysaccharides or fragments thereof can be of different serotypes.

[0067] After conjugation, the polysaccharide-polypeptide conjugate can be purified. For example, various chromatographic strategies can be used to purify the polysaccharide-polypeptide conjugate: SEC (using a column packed with Sepharose CL-6B), a combination of ion exchange chromatography (DEAE-Sepharose), and possibly HPSEC (using Sepharose CL-6B) and hydrophobic interaction chromatography (HIC) (using a column packed with Sephadex LH-20). For HPSEC purification on a Sepharose CL-6B column, a 4.0 mg quantity of a lyophilized mixture containing the polysaccharide-polypeptide conjugate, free polysaccharide, and free polypeptide is dissolved in 0.1 M formic acid buffer (ammonium salt) (pH 6.0) and applied to a column (60 cm x 4.0 cm) equilibrated with the same buffer at a flow rate of 0.5 mL / min. Isocratic elution can be performed using this buffer, and this process can be continuously monitored using a differential refractometer. Fractions can be collected, lyophilized, and used for further analysis. As a second strategy, 4.0 mg of the lyophilized mixture is dissolved in 0.1 M formic acid buffer (ammonium salt) (pH 6.0) and applied to a column (60 cm x 4.0 cm) equilibrated with the same buffer at a flow rate of 0.5 mL / min. A volume of the lyophilized reaction mixture can be dissolved in 500 μL of 0.1 M phosphate-buffered saline (PBS) (pH 7.0) and applied to a DEAE-Sepharose column previously equilibrated with the same buffer. The column flow-through can be collected, and after washing with buffer, elution can be performed using a gradient of NaCl (0.25-1.0 M). Each fraction can be analyzed for the presence of polysaccharides by the phenol-sulfuric acid technique. The eluted fractions can be pooled, lyophilized, resuspended in 0.1 M ammonium formate buffer (pH 6.0), and applied to a DEAE-Sepharose column previously equilibrated with the same buffer. The lyophilized reaction mixture can be applied to a CL-6B column. 5 mL fractions can be collected, dialyzed, lyophilized, and analyzed for protein and carbohydrate content. In a third chromatographic procedure, a 4.0 mg amount of the lyophilized reaction mixture can be dissolved in 1.0 mL of distilled water and applied to a column packed with Sephadex LH-20 previously equilibrated with distilled water at a flow rate of 0.5 mL / min. Elution can be carried out isocratically with distilled water, and the process can be monitored as described above. Fractions can be collected, lyophilized, and used for further analysis.

[0068] Additionally, "click" chemistry can be used to conjugate saccharides to polypeptides as disclosed in U.S. Pat. No. 8,753,645 and Hu et al., Towards the next generation of biomedicines by site selective conjugation, Chem. Soc. Rev., 2016, 45, 1691. "Click" chemistry can be used to conjugate modified CRMs with unnatural amino acids. 197 or CRM 197 can be conjugated to a polysaccharide. Pharmaceutical Composition

[0069] The saccharide-polypeptide conjugates described herein can be present in the form of a pharmaceutical composition. The saccharide-polypeptide conjugates described herein can be present in the form of a pharmaceutical composition in unit dosage form. The saccharide-polypeptide conjugates described herein can be present in the form of a vaccine. The pharmaceutical composition can comprise an immunogenic saccharide-polypeptide conjugate. The pharmaceutical composition can comprise an immunogenic capsular polysaccharide-polypeptide conjugate. The pharmaceutical composition can be present in unit dosage form and can comprise an immunogenic capsular polysaccharide-polypeptide conjugate. The pharmaceutical composition can comprise an immunogenic saccharide-polypeptide conjugate, where the capsular polysaccharide or immunogenic fragment thereof of the saccharide-polypeptide conjugate can be of one serotype. In another embodiment, the pharmaceutical composition can comprise an immunogenic saccharide-polypeptide conjugate, where the capsular polysaccharide or immunogenic fragment thereof can be from different serotypes, creating a mixture of different immunogenic saccharide-polypeptide conjugates. The pharmaceutical composition can comprise immunogenic saccharide-polypeptide conjugates, wherein the polypeptide is the same polypeptide in all of the immunogenic saccharide-polypeptide conjugates. In other embodiments, the pharmaceutical composition can comprise immunogenic saccharide-polypeptide conjugates, wherein the polypeptide of the saccharide-polypeptide conjugates is a mixture of different polypeptides.

[0070] For example, the pharmaceutical composition can comprise an immunogenic saccharide-polypeptide conjugate individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a specific S. pneumoniae selected from the group including 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise an immunogenic saccharide-polypeptide conjugate individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a specific S. pneumoniae selected from the group including 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of at least two immunogenic saccharide-polypeptide conjugates, each comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a specific S. pneumoniae selected from the group including 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of at least two immunogenic saccharide-polypeptide conjugates, each comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a specific S. pneumoniae selected from the group including 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of at least eight immunogenic saccharide-polypeptide conjugates, each comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a specific S. pneumoniae selected from the group including 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 34, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof derived from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not, or at least not, any one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; and the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition may comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof derived from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not, or at least not, any one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F; and the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof.The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise at least 10, 11, 12, 13, 14, 15, or 16 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition may comprise at least 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B).The pharmaceutical composition comprises at least 10, 11, 12, 13, 14, 15, or 16 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition can comprise at least 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38.The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B and 38. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 23A, 23B, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 23A, 23B, 24F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 23A, 23B, 24F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 23A, 24B, 35B, and 38.The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 23A, 23B, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 23A, 23B, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 23A, 23B, 24F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 23A, 24B, 35B, and 38. The pharmaceutical composition can include multiple saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 23A, 23B, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 23A, 23B, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 23A, 23B, and 33B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 20B, 23A, 23B, 34F, 31, 33B, 34, 35F, and 35B. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including at least 13 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including at least 15 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including at least 16 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B 31, 33F, 35B, 34, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 20B, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 2, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F, 22F, 23A, 23B, 24B, 31, 33F, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 1, 3, 4, 6A, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 24F, 33F, 35F, 35B, 38 or serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype selected from 1, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 23F, 24F, 33F, 35F, 35B, 38 or serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 13 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 15 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 34, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 16 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 2, 8, 9N, 10A, 11A, 12F, 15A, 15B, 16F, 17F, 22F, 23A, 23B, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype, including 1, 3, 4, 6A, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 24F, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 1, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 23F, 24F, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 3, 4, 7F, 8, 9N, 11A, 12F, 15A, 15B, 15C, 16F, 19A, 22F, 23A, 23B, and 35B.The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 1, 3, 4, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 19A, 19F, 22F, 23A, 23B, 23F, 24F, and 33F. The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 3, 4, 6B, 10A, 15A, 15B, 19A, 19F, 22F, 23F, 24F, 35B. The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least two additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least three additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least four additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least five additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least six additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least seven additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). and at least eight additional serotypes including serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least nine additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 10 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 11 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 12 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 34, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 13 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 34, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 14 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 15 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 16 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 17 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 18 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 19 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (which includes serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 20 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 21 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 22 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 34, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 23 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (which includes serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 24 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 34, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least 25 additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may be a combination of serogroups 6C, 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serogroups 20A and 20B), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 additional serotypes, including combinations thereof. The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least two additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least three additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least four additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least five additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may be used to treat serotypes 6C and 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, and 35B. The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least seven additional serotypes, including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least eight additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least nine additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 10 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 11 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 12 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 13 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 14 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 15 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 16 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 17 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 18 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 19 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 20 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 21 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 22 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 23 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 24 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may comprise a plurality of saccharide-polypeptide conjugates including serotype 6C and at least 25 additional serotypes including 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition may be a combination of serogroups 6C, 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serogroups 20A and 20B), and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 additional serotypes, including combinations thereof.The immunogenic saccharide-polypeptide conjugates can be present individually, each, or collectively in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the pharmaceutical composition. The immunogenic saccharide-polypeptide conjugates can include excipients, which can be present individually, each, or collectively in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the pharmaceutical composition. The immunogenic saccharide-polypeptide conjugate can include adjuvants, which can be present individually, each individually, or collectively in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the pharmaceutical composition. The pharmaceutical composition can be administered to one of a plurality of humans and elicits at least one of the following side effects in less than about 5%, 10%, 20%, or 30% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash.

[0071] In other embodiments, the pharmaceutical composition can include a saccharide-polypeptide conjugate, where the saccharide is derived from N. meningitidis. The pharmaceutical composition can include a saccharide-polypeptide conjugate, where the saccharide can be derived from S. enterica. The pharmaceutical composition can include a saccharide-polypeptide conjugate, where the saccharide can be derived from H. influenzae. The pharmaceutical composition can include a plurality of saccharide-polypeptide conjugates, including a saccharide from Neisseria meningitidis. The pharmaceutical composition can include a plurality of saccharide-polypeptide conjugates, including a saccharide from S. enterica. The pharmaceutical composition can include a plurality of saccharide-polypeptide conjugates, including a saccharide from H. influenzae.

[0072] The polypeptides of any of the multiple saccharide-polypeptide conjugates can be the same polypeptide. For example, the polypeptides of any of the multiple saccharide-polypeptide conjugates can be the same polypeptide. 197 The multiple saccharide-polypeptide conjugates can comprise a mixture of different polypeptides. For example, the multiple saccharide-polypeptide conjugates can comprise a mixture of CRMs. 197 , tetanus toxoid, diphtheria toxoid, or a mixture of any fragments thereof.

[0073] Any of the pharmaceutical compositions described herein can be in unit dosage form. Additionally, the pharmaceutical composition can further comprise an adjuvant. In some embodiments, the adjuvant can be an adjuvant containing aluminum cations. In other embodiments, the adjuvant can be aluminum phosphate, aluminum sulfate, or aluminum hydroxide. Further examples of adjuvants suitable for use in the pharmaceutical compositions include aluminum-based salts and variants thereof; emulsions (either water-in-oil or oil-in-water) and variants thereof, such as incomplete Freund's adjuvant, MF59® (Glaxosmithkline plc); PRR ligands and variants thereof, such as pathogen-associated molecular patterns (PAMPs); TLR3 and RLR ligands or variants thereof, such as synthetic analogs of dsRNA, poly(I:C); TLR4 ligands or variants thereof, such as bacterial flagellin, glucopyranosyl lipid adjuvant (GLA), monophosphoryl lipid A; TLR5 ligands or variants thereof, such as imiquimod, gardikimod, R848; TLR9 ligands or variants thereof, such as oxydeoxynucleotides containing CpG motifs (CpG oligonucleotides such as ODN1826 and ODN2006). ODN); NOD2 ligand or variants thereof, such as bacterial cell wall fragments (such as muramyl dipeptide [MDP]); saponin or variants thereof, including synthetic derivatives, such as QS21, TQL1055, or any combination of the above. The adjuvant can be 3M-052.

[0074] The pharmaceutical composition may further comprise an excipient, such as acacia, acesulfame potassium, acetic acid, glacial acetic acid, acetyl tributyl citrate, acetyl triethyl citrate, agar, albumin, alcohol, alginic acid, aliphatic polyesters, alitame, almond oil, alpha tocopherol, aluminum hydroxide adjuvant, aluminum oxide, aluminum phosphate adjuvant, aluminum stearate, ammonia solution, ammonium alginate, ascorbic acid, ascorbyl palmitate, aspartame, attapulgite, bentonite, or benzalkonium chloride. , benzethonium chloride, benzoic acid, benzyl alcohol, benzyl benzoate, boric acid, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, calcium alginate, calcium carbonate, dibasic calcium phosphate anhydrous, calcium phosphate dibasic dihydrate, tribasic calcium phosphate, calcium stearate, calcium sulfate, canola oil, carbomer, carbon dioxide, calcium carboxymethylcellulose, sodium carboxymethylcellulose, carrageenan, castor oil, hydrogenated castor oil, finely divided Crystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, cellulose acetate, cellulose acetate phthalate, ceratonia, cetostearyl alcohol, cetrimide, cetyl alcohol, cetylpyridinium chloride, chitosan, chlorhexidine, chlorobutanol, chlorocresol, chlorodifluoroethane (HCFC), chlorofluorocarbon (CFC), chloroxylenol, cholesterol, citric acid monohydrate, colloidal silicon dioxide, colorant, copovidone, corn oil, cottonseed oil, cresol, croscarmellose sodium, crospovidone Ingredients: cyclodextrin, cyclomethicone, denatonium benzoate, dextrates, dextrin, dextrose, dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethicone, dimethyl ether, dimethyl phthalate, dimethyl sulfoxide, dimethylacetamide, edetate disodium, docusate sodium, edetic acid, erythorbic acid, erythritol, ethyl acetate, ethyl lactate, ethyl maltol, ethyl oleate, ethyl vanillin, ethylcelluloseEthylene glycol palmitostearate, ethylene vinyl acetate, ethylparaben, fructose, fumaric acid, gelatin, liquid glucose, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, glyceryl palmitostearate, glycofurol, guar gum, hectorite, heptafluoropropane (HFC), hexetidine, hydrocarbon (HC), hydrochloric acid, hydroxyethyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, hydroxypropyl starch, hypromellose, hypromellose acetate succinate, hypromellose phthalate, imidurea, inulin, iron oxides, isomalt, isopropyl alcohol, isopropyl myristate, isopropyl palmitate P,almitate), Kaolin, Lactic Acid, Lactitol, Anhydrous Lactose, Lactose Monohydrate, Spray-Dried Lactose, Lanolin, Lanolin Alcohols, Hydrous Lanolin, Lauric Acid, Lecithin, Leucine, Linoleic Acid, Macrogol Hydroxystearate, Magnesium Aluminum Silicate Silicate), magnesium carbonate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, malic acid, maltitol, maltitol solution, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methylcellulose, methylparaben, mineral oil, light mineral oil, mineral oil and lanolin alcohols, monoethanolamine, monosodium glutamate, monothioglycerol, myristic acid, neohesperidin dihydrochalcone, nitrogen, nitrous oxide, octyldodecanol, oleic acid, oleyl alcohol, olive oil, palmitic acid, paraffin, peanut oil, pectin, petrolatum, petrolatum and lanolin alcohols, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phosphoric acid, polacrilin potassium, poloxamer, polycarbophil, polydextrose, polyethylene glycol, polyethylene oxidePolymethacrylates, poly(methyl vinyl ether / maleic anhydride), polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyvinyl acetate phthalate, polyvinyl alcohol, potassium alginate, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, potassium hydroxide, potassium metabisulfite, potassium sorbate, povidone, propionic acid, propyl gallate, propylene carbonate Ingredients: Propylene glycol, propylene glycol alginate, propylparaben, 2-pyrrolidone, raffinose, saccharin, saccharin sodium, saponite, sesame oil, shellac, simethicone, sodium acetate, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium borate, sodium chloride, sodium citrate dihydrate, sodium cyclamate, sodium hyaluronate, sodium hydroxide, sodium lactate, sodium lauryl sulfate, sodium disulfite Sodium, dibasic sodium phosphate, monobasic sodium phosphate, sodium propionate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty acid esters), sorbitol, soybean oil, starch, pregelatinized starch, sterilizable corn starch, stearic acid, stearyl alcohol, sucralose, sucrose, compressible sugar, powdered sugar, granulated sugar, sulfobutyl ether b-cyclodextrin, sulfuric acid, sunflower oil, hard The emulsifying agent may be a fat, a suppository base, talc, tartaric acid, tetrafluoroethane (HFC), thaumatin, thimerosal, thymol, titanium dioxide, tragacanth, trehalose, triacetin, tributyl citrate, triethanolamine, triethyl citrate, vanillin, hydrogenated vegetable oil, water, anionic emulsifying wax, carnauba wax, cetyl ester wax, microcrystalline wax, nonionic emulsifying wax, white wax, yellow wax, xanthan gum, xylitol, zein, zinc acetate, or zinc stearate.

[0075] The pharmaceutical composition may further comprise a surfactant or an emulsifier, or a combination thereof. The surfactant may be a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. In some embodiments, the polysorbate may be polysorbate 80. In some embodiments, the polymeric glycol may be polyethylene glycol. The surfactant may be TWEEN. The pharmaceutical composition may comprise a polyethylene glycol specific for its molecular weight. The pharmaceutical composition may comprise a preservative. The pharmaceutical composition may comprise a salt. The salt is an inorganic salt. The pharmaceutical composition may further comprise an antifungal compound or a salt thereof. The antifungal compound may be thimerosal, phenol, benzethonium chloride, or 2-phenoxyethanol. The pharmaceutical composition may further comprise a chelating agent. The chelating agent may be ethylenediaminetetraacetic acid (EDTA). The pharmaceutical composition may further comprise a buffer. The buffer may be succinic acid buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, or potassium phosphate buffer. The pharmaceutical composition may further comprise sterile water. When administered intramuscularly to a human, the pharmaceutical composition may induce an immune response lasting from about 1 week to about 30 years. The pharmaceutical composition may retain at least about 60% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container may retain at least about 70% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container may retain at least about 70% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container may retain at least about 70% of its original biological activity when stored in a sealed container at a temperature of about 50% relative humidity and 1 atm atmospheric pressure. The pharmaceutical composition can retain at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and when the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm.The pharmaceutical composition can retain at least about 90% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and when the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm.

[0076] The polysaccharide-polypeptide conjugates described herein can be adjuvanted. Suitable adjuvants include aluminum hydroxide gel (alum) or aluminum phosphate, but can also be calcium, magnesium, iron, or zinc salts, or insoluble suspensions of acylated tyrosine, acylated sugars, cationic or anionic derivatized polysaccharides, or polyphophazenes. The adjuvant can be 3M-052. When adjuvanted with an aluminum salt, the ratio of aluminum salt to polysaccharide can be less than 10:1 (w / w), less than 8:1 (w / w), or more than 2:1 (w / w) in the vaccine dose or formulation.

[0077] The amount of polypeptide in a vaccine dose can be selected to induce an immunoprotective response without significant adverse events. A dose or formulation can contain 0.1-50 μg of polysaccharide, as 0.1-100 μg of polysaccharide and / or polysaccharide-polypeptide conjugate. A polysaccharide-polypeptide conjugate can contain 1-10 μg of polysaccharide, 1-5 μg of polysaccharide, or 2-5 μg of polysaccharide. The polypeptide content in a vaccine can be within the range of 1-100 μg of polypeptide, 5-50 μg of polypeptide, or 5-25 μg of polypeptide.

[0078] Vaccine dosages can range from 1 to 5 μg of polysaccharide for polysaccharide-polypeptide conjugate delivered intramuscularly as a single 0.5 mL injection. Vaccine dosages can include aluminum phosphate (1 mg / mL) and 0.01% polysorbate 80. How to use

[0079] The saccharide-polypeptide conjugates described herein can be formulated for use in vaccines. Pharmaceutical compositions comprising the saccharide-polypeptide conjugates described herein can be formulated for use in vaccines. Pharmaceutical compositions comprising the saccharide-polypeptide conjugates described herein can be used in vaccines. The vaccines can be administered to a subject. The vaccines can be used as prophylactics against diseases caused by S. pneumoniae, such as meningitis, pneumonia, and serious invasive diseases associated with pneumococcal infection. The subject can be a human. The subject or human can be a subject or human in need thereof.

[0080] The saccharide-polypeptide conjugate can be administered by intramuscular injection. The saccharide-polypeptide conjugate can be administered by intramuscular, intraperitoneal, intradermal, or subcutaneous routes, or by oral / gastrointestinal, respiratory, or genitourinary mucosal administration. The saccharide-polypeptide conjugate can be administered by suppository. The saccharide-polypeptide conjugate can be administered intranasally. The saccharide-polypeptide conjugate can induce an opsonization response after administration. The saccharide-polypeptide conjugate can induce the production of antibodies against the serotype of the saccharide-polypeptide conjugate. This antibody response can be measured by an in vitro opsonization assay after administration of the saccharide-polypeptide conjugate.

[0081] Pharmaceutical compositions containing saccharide-polypeptide conjugates can be administered by intramuscular injection. Pharmaceutical compositions containing saccharide-polypeptide conjugates can be administered intramuscularly, intraperitoneally, intradermally, or subcutaneously, or via oral / gastrointestinal, respiratory, or genitourinary mucosal administration. Pharmaceutical compositions containing saccharide-polypeptide conjugates can be administered intranasally. Pharmaceutical compositions can be administered intramuscularly and can elicit an immune response lasting from one week to 30 years. Saccharide-polypeptide conjugates can elicit an opsonization response after administration. Pharmaceutical compositions containing saccharide-polypeptide conjugates can elicit the production of antibodies against the serotype of the saccharide-polypeptide conjugate. This antibody response can be measured by an in vitro opsonization assay after administration of a pharmaceutical composition containing a saccharide-polypeptide conjugate.

[0082] For example, a pharmaceutical composition administered to a subject can include a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof derived from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F, or is not at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F. or not 23F, the serotype is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, or 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least nine serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 10 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 11 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition administered to a subject can comprise at least 12 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 13 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 14 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 15 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 16 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 17 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition administered to a subject can comprise at least 18 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 19 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 20 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can comprise at least 21 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 22 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B).For example, a pharmaceutical composition administered to a subject can include a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof derived from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F, or is not at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F. or 23F, the serotype is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, or 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least nine serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 10 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 11 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition administered to the subject can comprise at least 12 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 13 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 14 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 15 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 16 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 17 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition administered to the subject can comprise at least 18 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 19 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 20 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise at least 21 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical compositions administered to the subject are 2, 6C, 8, 9N, 1. The conjugates may comprise at least 22 serotypes selected from the group consisting of 0A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B). The pharmaceutical composition administered to the subject may comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to a subject can include multiple saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38.The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B and 38. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 13 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof.The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 14 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 15 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 16 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 17 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof.The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 18 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject may be selected from the group consisting of serogroups 6C, 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (serotypes 20A and 20B). and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 additional serotypes (including The pharmaceutical composition administered to the subject may comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 additional serotypes, including 1, 3, 4, 6A, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 24F, 24B, 33F, 34, 35F, 35B, 38 or serogroup 20 (including serotypes 20A and 20B).The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 additional serotypes 1, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 23F, 24F, 24B, 33F, 34, 35F, 35B, 38 or serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 13 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 14 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 15 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof.The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 16 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 17 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates, including at least 18 of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The pharmaceutical composition administered to the subject may be a combination of serotypes 6C and 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 1 3F, 34, 35F, 35B, 38, and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 additional serotypes including serogroup 3F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or combinations thereof. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 additional serotypes, including 1, 3, 4, 6A, 6B, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 24F, 24B, 33F, 34, 35F, 35B, 38, or serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 additional serotypes 1, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 23F, 24F, 24B, 33F, 34, 35F, 35B, 38, or serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can include a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B.The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional serotypes selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 34, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 additional serotypes selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, 34, and 35B. The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 additional serotypes selected from 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B). The pharmaceutical composition administered to the subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 3, 4, 7F, 8, 9N, 11A, 12F, 15A, 15B, 15C, 16F, 19A, 22F, 23A, 23B, and 35B.A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 1, 3, 4, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 19A, 19F, 22F, 23A, 23B, 23F, 24F, and 33F. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 3, 4, 6B, 10A, 15A, 15B, 19A, 19F, 22F, 23F, 24F, and 35B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 9N, 15A, 15C, 16F, 23A, 23B, and 33B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 15A, 15C, 16F, 23A, 23B, and 33B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. A pharmaceutical composition administered to a subject can comprise a plurality of saccharide-polypeptide conjugates comprising serotype 6C and at least one additional serotype selected from 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. The pharmaceutical composition administered to the subject may comprise a plurality of saccharide-polypeptide conjugates, including serotype 6C and at least one additional serotype selected from 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B.

[0083] Each dosage can be formulated to 0.5 mL. The formulation can include normal saline. The formulation can have a pH of 6.5. The formulation can have a pH of 6.3 to 6.7 or 6.2 to 6.8. The formulation can have a pH of 5.8. The formulation can have a pH of 5.5 to 6.1 or 5.6 to 6.0. The formulation can include 0.001% to 0.01% polysorbate 80.

[0084] Two different pharmaceutical compositions can be administered to a subject. The first pharmaceutical composition can be administered to a subject at least about 4 weeks, 6 weeks, 2 months, 6 months, 8 months, 1 year, 2 years, or 5 years before, or at least about 4 weeks, 6 weeks, 2 months, 6 months, 8 months, 1 year, 2 years, or 5 years after, administration of the second pharmaceutical composition to the subject. The first pharmaceutical composition can be administered to a subject at least 4 weeks, 6 weeks, 12 weeks, 24 weeks, 6 months, 8 months, 10 months, 12 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, or older. The first pharmaceutical composition can be administered to a subject aged between 4 weeks and 2 years. The first pharmaceutical composition can be administered to a subject aged between 18 and 100 years. The first pharmaceutical composition can be administered to a subject aged 100 years or older. In some embodiments, the second pharmaceutical composition can comprise a saccharide-polypeptide conjugate comprising a polypeptide having at least about 70%, 80%, 90%, 95%, 99%, or 100% length homology, about 70%, 80%, 90%, 95%, 99%, or 100% sequence homology, or a combination thereof, to the polypeptide of the saccharide-polypeptide conjugate of the first pharmaceutical composition. For example, the polypeptide of the saccharide-polypeptide conjugate of the first pharmaceutical composition and the polypeptide of the saccharide-conjugate of the second pharmaceutical composition can be a CRM. 197In other embodiments, the second pharmaceutical composition can comprise a saccharide-polypeptide conjugate that comprises a polypeptide having less than 70% length homology, less than 70% sequence homology, or a combination thereof, to the polypeptide of the saccharide-polypeptide conjugate of the first pharmaceutical composition.

[0085] In some embodiments, the first pharmaceutical composition may comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not at least 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F, and the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B). The first pharmaceutical composition can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (which includes serotypes 20A and 20B).In some embodiments, the first pharmaceutical composition may comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof from a serotype of S. pneumoniae conjugated to a polypeptide, wherein the serotype is not at least 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F, and the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B), or a combination thereof. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B). The first pharmaceutical composition can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including 20A and 20B). The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can include a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B.The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 23A, 23B, and 33B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. The first pharmaceutical composition can include a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B.The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. In other embodiments, the first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including a serotype that is at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The first pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. As noted above, when any of the first pharmaceutical compositions is administered, the first pharmaceutical composition can be separate from the second pharmaceutical composition administered to the subject.

[0086] In some embodiments, the second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotypes that are at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates comprising serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including a serotype that is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B), wherein the serotype of the first pharmaceutical composition and the serotype of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including a serotype that is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including 20A and 20B), wherein the serotype of the first pharmaceutical composition and the serotype of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof from a serotype of S. pneumoniae conjugated to a polypeptide, the serotype being at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 46A, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1109, 1111, , 12, or 13, the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), and the serotype of the first pharmaceutical composition and the serotype of the second pharmaceutical composition are distinct.The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B), wherein the serotypes of the first pharmaceutical composition and the serotypes of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (which includes serotypes 20A and 20B), wherein the serotypes of the first pharmaceutical composition and the serotypes of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a saccharide-polypeptide conjugate comprising a capsular polysaccharide or immunogenic fragment thereof from a serotype of S. pneumoniae conjugated to a polypeptide, the serotype being at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 46A, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1109, 1111, , 12, or 13, the serotype is at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38 and serogroup 20 (including 20A and 20B), and the serotype of the first pharmaceutical composition and the serotype of the second pharmaceutical composition are distinct.The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including 20A and 20B), wherein the serotypes of the first pharmaceutical composition and the serotypes of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 serotypes selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including 20A and 20B), wherein the serotypes of the first pharmaceutical composition and the serotypes of the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct.The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B, where the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 23A, 23B, and 33B, where the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B, where the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct.The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B and 38, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct. The second pharmaceutical composition can comprise a plurality of saccharide-polypeptide conjugates, including serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B, wherein the serotypes of the first pharmaceutical composition and the second pharmaceutical composition are distinct.

[0087] In other embodiments, the first and second pharmaceutical compositions can be administered at least about 4 weeks apart, at least about 6 weeks apart, at least about 2 months apart, at least about 3 months apart, at least about 4 months apart, at least about 5 months apart, at least 6 months apart, at least about 7 months apart, at least about 8 months apart, at least about 9 months apart, at least about 10 months apart, at least about 11 months apart, at least about 1 year apart, at least about 1.5 years apart, at least about 2 years apart, at least about 5 years apart, at least about 10 years apart, at least about 15 years apart, at least about 20 years apart, or at least about 30 years apart.

[0088] In some embodiments, the serotype of the first pharmaceutical composition may be derived from a different bacterium than the serotype of the second pharmaceutical composition. For example, the serotype of the first composition may be derived from Streptococcus pneumoniae, and the serotype of the second composition may be derived from Neisseria meningitidis, Salmonella enterica, or Haemophilus influenzae. The serotype of the first pharmaceutical composition may be derived from Neisseria meningitidis, Salmonella enterica, or Haemophilus influenzae, and the serotype of the second pharmaceutical composition may be derived from Streptococcus pneumoniae.

[0089] In some embodiments, the serotype of the first pharmaceutical composition can be derived from the same bacterium as the serotype of the second pharmaceutical composition. For example, the first pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F 38, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38 and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae.The first pharmaceutical composition can include at least one of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, 38, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae.The first pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae. The first pharmaceutical composition can include at least one of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae, and the second pharmaceutical composition can include at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae.The first pharmaceutical composition can include serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae. The first pharmaceutical composition can include serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae.The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 6C, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae. The first pharmaceutical composition can include serotypes 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae.The first pharmaceutical composition can include serotypes 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae. and the second pharmaceutical composition can comprise serotypes 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae, and The first pharmaceutical composition can include serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second pharmaceutical composition can include serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae. The first pharmaceutical composition can comprise serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 35B, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae, and the second pharmaceutical composition can comprise serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae.

[0090] Different serotypes of S. pneumoniae may be more prevalent or endemic in different geographic regions. For example, a particular group of serotypes may commonly cause pneumococcal disease in the United States and Canada. The serotypes in this group may differ from those in Europe, the Middle East, North Africa, Japan, and Korea. For example, administering to a subject a pharmaceutical composition containing multiple saccharide-polypeptide conjugates, including serotypes 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 19F, 19A, 20A, 22F, 23A, 23B, 33F, 35F, 35B, and 38, can improve protection against S. pneumoniae disease in subjects in the United States and / or Canada. Protection against S. pneumoniae disease in subjects in the United States and / or Canada can be improved by administering to the subject a pharmaceutical composition comprising a plurality of saccharide-polypeptide conjugates, including serotypes 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 33F, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B). Protection against S. pneumoniae disease in subjects in Europe, the Middle East, and / or North Africa can be improved by administering to a subject a pharmaceutical composition comprising a plurality of saccharide-polypeptide conjugates, including serotypes 1, 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19F, 19A, 22F, 23F, 23A, 23B, 24F, 33F, 35F, 35B, and 38. Protection against S. pneumoniae disease in Japanese and / or Korean subjects can be improved by administering to the subject a pharmaceutical composition comprising multiple saccharide-polypeptide conjugates, including serotypes 3, 4, 6A, 6B, 6C, 7F, 10A, 11A, 14, 15A, 15B, 15C, 19F, 19A, 22F, 23F, 23A, 24F, 35F, 35B, and 38. Furthermore, invasive clinical disease can be caused by serotype 20B (rather than serotype 20A).The subject may be a human residing in a particular geographic location, as indicated above, or may be a human visiting a particular geographic location, as indicated above.

[0091] The method of administration can include administering to the subject a first saccharide-polypeptide conjugate composition, and at least four weeks after administering the first saccharide-polypeptide conjugate composition, administering to the subject a second saccharide-polypeptide conjugate composition, wherein the first saccharide-polypeptide conjugate composition comprises a first polypeptide having at least about 70%, 80%, 90%, 95%, 99%, or 100% length identity, about 70%, 80%, 90%, 95%, 99%, or 100% sequence identity, or a combination thereof, to a second polypeptide of the second saccharide-polypeptide conjugate composition, and wherein the first saccharide-polypeptide conjugate composition comprises a first serotype and the second saccharide-polypeptide conjugate composition comprises a second serotype, wherein the first serotype and the second serotype are different. In some embodiments, the first serotype is derived from the same bacterium as the second serotype. For example, the saccharide-conjugated polypeptide of the first pharmaceutical composition and the saccharide-conjugated polypeptide of the second pharmaceutical composition may be CRMs. 197 In other embodiments, the second pharmaceutical composition comprises a saccharide-polypeptide conjugate comprising a polypeptide having less than about 70% length homology, less than about 70% sequence homology, or a combination thereof, to the polypeptide of the saccharide-polypeptide conjugate of the first pharmaceutical composition.

[0092] In other embodiments, performing any of the methods described herein may not result in more than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% suppression of the immunity conveyed by the first vaccine containing the immunogenic saccharide-polypeptide conjugate, as measured by an antibody response to the saccharide of the immunogenic saccharide-polypeptide conjugate, where the antibody response is measured by measuring antibody titers. Saccharide-Polypeptide Conjugate Kits

[0093] The saccharide-polypeptide conjugates and pharmaceutical compositions described herein can be contained in a kit. The kit can further include instructions for use. The saccharide-polypeptide conjugates and / or pharmaceutical compositions can be stored in a container. The container can be a syringe. The container can be a vial. The vial can be a 2 mL borosilicate glass vial. The vial can be a 3 mL non-siliconized USP Type I glass vial. The top can be an aluminum crimp top. The container can be a vial with a rubber stopper or a stopper made of a flexible reclosable material. The container can be a stoppered vial. The stopper can be a non-siliconized stopper. The vial can have a fill volume of 0.6 mL. The extractable volume can be 0.5 mL. [Example]

[0094] The following examples are included to further illustrate some aspects of the disclosure and should not be used to limit the scope of the embodiments described herein. Example 1 How to establish a cell bank

[0095] This example shows the polysaccharide-CRM 197Establishment of a cell bank for S. pneumoniae strains for use in producing polysaccharides for conjugate vaccines is shown below. Any strain producing a polysaccharide listed in Table 1 or Figures 11A-11F (i.e., polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, or 38 and serogroup 20 (including serotypes 20A and 20B)) can be established as a cell bank by this method. S. pneumoniae strains are provided by either the Centers for Disease Control and Prevention (CDC) (USA) or the Culture Collection, University of Gothenburg (CCUG). The cell banking process is shown in Figure 2. A vial of S. pneumoniae strain is inoculated and then grown overnight on an agar plate. A single colony is selected from this plate and streaked onto a new agar plate for overnight growth. This process is repeated three times to purify the strain before inoculating and incubating the flask. After the inoculated flask has grown for a sufficient time, the contents of the flask are harvested by centrifugation. The product of the centrifugation is resuspended in approximately 20% glycerol medium and then aliquoted into cryogenic vials. The cryogenic vials contain the cell bank and are stored at -70°C until use.

[0096] A S. pneumoniae strain producing serotype 6C polysaccharide was acquired by the University of Alabama, Birmingham (UAB), and a 6C cell bank was established. The cell banking process is shown in Figure 2. A vial of S. pneumoniae strain was inoculated with the 6C polysaccharide serotype and then grown overnight on an agar plate. A single colony was selected from this plate and streaked onto a new agar plate for overnight growth. This process was repeated three times to purify the strain before inoculating and incubating the flask. After the seed flask had grown for a sufficient time, the contents of the flask were harvested by centrifugation. The centrifugation product was resuspended in approximately 20% glycerol medium and then aliquoted into cryogenic vials. The cryogenic vials contained the cell bank for each strain and were stored at -70°C until use.

[0097] S. pneumoniae strains producing serotype 8 polysaccharide, serotype 10A polysaccharide, serotype 11A polysaccharide, serotype 12F polysaccharide, serotype 22F polysaccharide, or serotype 33F polysaccharide were acquired by either the Centers for Disease Control and Prevention (CDC) (USA) or the Gothenburg University Culture Collection (CCUG) to establish cell banks. The cell banking process is shown in Figure 2. For each polysaccharide serotype, a vial of S. pneumoniae strain was inoculated and then grown overnight on an agar plate. A single colony was selected from this plate and streaked onto a new agar plate for overnight growth. This process was repeated three times to purify the strain before inoculating and incubating the flask. After the inoculated flask was grown for a sufficient time, the contents of the flask were harvested by centrifugation. The product of the centrifugation was resuspended in approximately 20% glycerol medium and then dispensed into cryogenic vials. Cell banks for each strain were contained in cryogenic vials and stored at −70°C until use. Example 2 Methods for isolating and purifying polysaccharides

[0098] This example shows the polysaccharide-CRM197 A method for isolating and purifying polysaccharides from cell banks for use in conjugate vaccines is described. Any of the polysaccharides listed in Table 1 or Figures 11A-11F (i.e., polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, or 38 and serogroup 20 (including serotypes 20A and 20B)) are isolated and purified by this method. The process for polysaccharide isolation and purification is shown in Figure 4. A vial of the cell bank generated as described in Example 1 is used to inoculate a flask containing the appropriate culture medium and incubated overnight. This culture is used to inoculate a new culture for fermentation in a fermentor. The culture is grown under conditions appropriate for the strain. After fermentation, the cells are inactivated and then treated with deoxycholate to rupture the cells and release the capsular polysaccharide. The cell-free broth is then harvested by centrifugation to remove debris and a depth filtration step through a 0.45 μm filter. The product is then purified and the polysaccharide size is determined. Quality control tests on the product are performed, including purity assays and confirmation of strain inactivation (no growth after plating).

[0099] A cell bank of S. pneumoniae strains producing serotype 8, serotype 10A, serotype 11A, serotype 12F, serotype 22F, or serotype 33F was used to inoculate a flask containing the appropriate culture medium and incubated overnight. This culture was used to inoculate a new culture for fermentation in a fermentor. This culture was grown under conditions appropriate for that particular strain. After fermentation, the cells were inactivated and then treated with deoxycholate to rupture the cells and release the capsular polysaccharide. The cell-free broth was then harvested after a depth filtration step through a 0.45 μm filter to remove debris. The product was then purified, and the polysaccharide size was determined. Quality control tests on the product, including purity assays and confirmation of strain inactivation (no growth after plating), were performed. Example 3 CRM 197 How to generate

[0100] This example demonstrates the use of CRMs for use in saccharide-polypeptide vaccines. 197 The production of (SEQ ID NO: 1) is shown in Figure 3. 197 Vials from the working cell bank were inoculated into flasks containing the appropriate culture medium and incubated overnight. The fermentation culture was centrifuged, the contents harvested, clarified, and then filtered through a 0.2 μm filter. The filtrate was concentrated approximately 10-fold and then diafiltered to exchange the buffer and remove contaminants. The protein was further purified by microfiltration using a membrane of appropriate pore size. The filtrate was then further purified by anion exchange chromatography, followed by ammonium sulfate precipitation and filtration (0.45 μm). Final purification using hydrophobic chromatography yielded highly purified CRM. 197 The protein was concentrated and diafiltered, then filtered through a 0.2 μm filter. The product was stored at −70° C. until use in conjunction with any of the polysaccharides listed in Table 1 or Figures 11A-11F (i.e., polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B)). Quality control tests were performed on the product to determine concentration (BCA to Lowry NLT 15 mg / mL), purity (SDS-PAGE gel for target purity ≥ 95%), CRM 197 were assessed for confirmation of identity (using monoclonal antibodies), size assessment (SDS-Page for a target of approximately 58 kDa), endotoxin level assessment (LAL kit targeting less than 1 IU / μg of protein), lack of toxicity assessment (Vero cytotoxicity or DT enzyme test; nonlethal in guinea pigs), pH assessment (meter, targeting 6.5 ± 0.2), and appearance assessment (visual targeting clear liquid). Example 4 Conjugation Methods

[0101] This example demonstrates the conjugation of capsular polysaccharides to polypeptides via CDAP conjugation chemistry. The polysaccharides are isolated and thawed as described in Example 2. The polypeptides are selected from any of the following: diphtheria and tetanus toxoids, and variants thereof (e.g., DT, CRM). 197 , TT), cholera toxoid, pertussis toxoid, inactivated or mutated pneumococcal pneumolysin, pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or B streptococcus, noncapsulated H. influenzae P4 protein, noncapsulated H. influenzae P6 protein, M. catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, or any fragment thereof. The polysaccharide is activated by CDAP chemistry in a CDAP solution. The polypeptide is added to the activated polysaccharide, and the conjugation reaction is then quenched. The reaction mixture is filtered, diafiltered, and concentrated. The polysaccharide-polypeptide conjugate is then separated using gel filtration chromatography. These conjugates are then concentrated to the final polysaccharide-polypeptide conjugate. Example 5 Polysaccharide-CRM 197 Conjugation Methods

[0102] This example demonstrates the use of CRMs of capsular polysaccharides via CDAP conjugation chemistry. 197The isolated polysaccharide can be any of the polysaccharides listed in Table 1 or Figures 11A-11F (i.e., polysaccharides from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B)), or any of the polysaccharides listed in Example 2, and can be conjugated to CRM 197 is described in Example 3. The conjugation process is shown in Figure 5. Polysaccharide and CRM 197 The polysaccharide is mixed with the CDAP solution and then incubated at an appropriate temperature for polysaccharide activation. 197 The calculated amount of glycine or any other suitable quenching reagent is then added to the activated polysaccharide. The conjugation reaction mixture is then filtered, diafiltered, and concentrated to give the polysaccharide-CRM 197 The polysaccharide-CRM conjugate is then 197 The conjugate is purified by gel filtration chromatography. 197 The fractions containing the conjugate are pooled, concentrated, and then filtered through a 0.2 μm filter. Store the product at 2-8 °C.

[0103] Isolated polysaccharides from serotype 8, serotype 10A, serotype 11A, serotype 12F, serotype 22F, or serotype 33F as described in Table 1 and Example 2, and CRM as described in Example 3 197 Polysaccharides from serotype 8, serotype 10A, serotype 11A, serotype 12F, serotype 22F, or serotype 33F were mixed with the CDAP solution and then incubated at an appropriate temperature for polysaccharide activation. 197 The calculated amount of activated polysaccharide was then added. The reaction was then quenched using glycine or any other suitable quenching reagent. The conjugation reaction mixture was filtered, diafiltered, and concentrated to give the polysaccharide-CRM. 197The polysaccharide-CRM conjugate was then prepared. 197 The conjugate was purified by gel filtration chromatography. 197 The fractions containing the conjugate were pooled, concentrated, and then filtered through a 0.2 μm filter. The product was stored at 2-8°C. Example 6 Polysaccharide-CRM in vaccine compositions 197 Conjugate combinations

[0104] This example shows the polysaccharide-CRM 197 The polysaccharide combinations used in the conjugate vaccine are shown. The serotype of each polysaccharide is CRM 197 The polysaccharide-CRM197 conjugates were individually conjugated to each serotype. 197 The conjugates are combined into vaccine compositions. Exemplary Polysaccharide-CRM 197 See Table 2 below for conjugate serotype combinations. Table 2. Polysaccharide-CRM 197 Polysaccharide combinations in conjugate vaccines [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] Example 7 Polysaccharide-CRM 197 Conjugate vaccine formulation

[0105] This example shows the polysaccharide-CRM 197 Formulation as a conjugate vaccine. Polysaccharide-CRM from Example 5. 197 Any combination of conjugates, or the combinations listed in Example 6, are used in the vaccine composition. Polysaccharide-CRM197 In addition to the conjugate, the vaccine is composed of normal saline and 0.01% polysorbate 80 with a pH of 6.5±0.2. 197 This formulation of vaccine composition is stored in 2 mL glass vials with aluminum crimp tops and stoppers at 2-8° C. Vials are filled to a volume of 0.60 mL, leaving an extractable volume of 0.50 mL. Example 8 Polysaccharide-CRM 197 Process for the formulation of conjugate vaccines

[0106] This example shows the polysaccharide CRM 197 Illustrates the formulation process for conjugate vaccines. Polysaccharide CRM 197 The conjugates are prepared as described in the examples above. The formulation process is shown in Figure 6. 0.9% w / v sterile saline is added to the formulation vessel. Each type of conjugate is sterilized separately and an appropriate volume of each monovalent bulk polysaccharide-CRM is added. 197 Add the conjugate one at a time to the formulation vessel (volume is relative to polysaccharide concentration). Adjust the pH to 6.5±0.2 using 1N HCl or 1N NaOH. Optionally, add the polysaccharide-CRM 197 An adjuvant, such as aluminum phosphate, is added to the conjugate mixture to a final concentration of 1 mg / mL. A calculated volume of sterile saline is added to the appropriate final concentration, and the mixture is then blended for approximately 2 hours. The formulation in the bulk container is then added to a filling machine, which fills, stoppers, and seals the final vaccine product vials. These vials are then inspected, labeled, and packaged for distribution. During this process, the pH, appearance, and sterility of the vaccine are monitored. During the filling, stoppering, and sealing steps, the appearance, pH, polysaccharide identity, CRM 197 The CRMs for each serotype are evaluated for identity, sterility, saccharide content, total protein content, endotoxin content, pyrogen content, general safety, osmolality, and extractable volume. 197For conjugates, 1 to 5 micrograms of polysaccharide is contained in 0.5 mL of vaccine in a vial. Example 9 Rabbit Toxicological Safety Study

[0107] This example describes rabbit toxicology studies used to evaluate vaccine safety. Polysaccharide-polypeptide conjugates were produced according to Examples 1, 2, 3, and 5 above, and derived from serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B, and were used in the CRM 197 Alternatively, polysaccharide-polypeptide conjugates may be produced according to Examples 1, 2, 3, and 5 above, and derived from serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B, and conjugated to CRM 197 These polysaccharide-CRMs contain polysaccharides from S. pneumoniae that are conjugated to 197 The conjugate is used in a vaccine formulated according to Examples 6 and 7. This polysaccharide-CRM 197 The conjugate vaccine will be administered intramuscularly to 5-month-old rabbits (10 per sex) at a dose of 0.5 ml per injection into the right thigh muscle on days 1, 22, 43, 64, and 85. 0.5 ml of 0.9% sodium chloride will be injected into the control group (10 per sex). 0.125 mg of aluminum phosphate as adjuvant will be injected into the AlP04 group (10 per sex). Five animals per sex per group will be allowed a 4-week treatment-free recovery period after the last injection. Treatment-related mortality and any other toxicologically relevant changes in relative food intake, ophthalmoscopic parameters, heart rate, respiratory rate, gross morphology, or organ weights will be assessed to confirm the lack of systemic toxicity. Injection sites will be analyzed for reactions and any chronic inflammation. Polysaccharide CRM 197 No serum antibodies specific for each polysaccharide in the conjugate vaccine were observed in the test group.

[0108] Polysaccharide-polypeptide conjugates were produced according to Examples 1, 2, 3, and 5 above, and were derived from serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B, and were CRM 197 Alternatively, polysaccharide-polypeptide conjugates may be produced according to Examples 1, 2, 3, and 5 above, and derived from serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B, and conjugated to CRM 197 These polysaccharide-CRMs contain polysaccharides from S. pneumoniae that are conjugated to 197 The conjugate is used in a vaccine formulated according to Examples 6 and 7. This polysaccharide CRM 197 The conjugate vaccine, with or without aluminum phosphate adjuvant, is administered intramuscularly as a single 0.5 mL injection into the left thigh muscle of 5-month-old male rabbits, three per group. 0.5 mL of 0.9% sodium chloride is administered to a control group (3 males). Treatment-related mortality, changes in body weight, relative food intake, or treatment-related macroscopic or microscopic lesions are assessed. Example 10 Vaccine Compositions and Administration Methods

[0109] This example demonstrates a vaccine composition and a method for administering the vaccine composition. The aqueous vaccine composition contains diphtheria and tetanus toxoids and their variants (e.g., DT, CRM). 197, TT), cholera toxoid, pertussis toxoid, inactivated or mutated pneumococcal pneumolysin, pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, or a fragment of any thereof; or any combination of the polysaccharide-polypeptide conjugates described in Example 6. The aqueous vaccine is formulated to contain 1-4 micrograms of each serotype of polysaccharide for the polysaccharide-polypeptide conjugate, aluminum phosphate (1 mg / mL), and 0.01% polysorbate 80 in a single 0.5 mL injection delivered intramuscularly to a subject. The vaccine is administered to children between 4 weeks and 2 years of age. The vaccine is administered to children between 2 and 18 years of age. The vaccine is administered to adults at least 50 years of age. Example 11 Vaccination for protection against epidemic S. pneumoniae serotypes in the United States and Canada

[0110] This example illustrates vaccination of a subject to provide protection against S. pneumoniae for subjects residing in or visiting the United States and / or Canada. The aqueous vaccine composition is composed of an immunogenic saccharide-polypeptide conjugate comprising capsular polysaccharides from S. pneumoniae serotypes 3, 4, 6A, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 19F, 19A, 22F, 23A, 23B, 33F, 35F, 35B, 38 and serogroup 20 (including serotypes 20A and 20B), e.g., serotypes 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. The aqueous vaccine is formulated to contain 1-5 micrograms of each serotype and aluminum phosphate as an adjuvant. A single 0.5 mL dose of the aqueous vaccine is given to subjects via intramuscular injection. Following administration, the vaccine provides improved immunity to endemic S. pneumoniae disease in the United States and Canada. Example 12 Vaccination for protection against S. pneumoniae serotypes prevalent in Europe, the Middle East, and North Africa

[0111] This example demonstrates a vaccine composition that provides protection against S. pneumoniae for subjects residing in or visiting Europe, the Middle East, and / or North Africa. The aqueous vaccine composition is composed of an immunogenic saccharide-polypeptide conjugate comprising capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19F, 19A, 22F, 23F, 23A, 23B, 24F, 33F, 35F, 35B, and 38, e.g., serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35F. The aqueous vaccine is formulated to contain 1-5 micrograms of each serotype and aluminum phosphate as an adjuvant. A single 0.5 mL dose of the aqueous vaccine is administered intramuscularly to subjects. Following administration, subjects are immunized against S. pneumoniae disease endemic in Europe, the Middle East, and North Africa. Example 13 Vaccination for protection against prevalent S. pneumoniae serotypes in Japan and Korea

[0112] This example demonstrates a vaccine composition that provides protection against S. pneumoniae in subjects residing in or visiting Japan and / or South Korea. The aqueous vaccine composition is composed of an immunogenic saccharide-polypeptide conjugate containing capsular polysaccharides from S. pneumoniae serotypes 3, 4, 6A, 6B, 6C, 7F, 10A, 11A, 14, 15A, 15B, 19F, 19A, 22F, 23F, 23A, 24F, 35F, 35B, and 38, e.g., serotypes 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38. The aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant. A single 0.5 mL dose of the aqueous vaccine is administered to subjects via intramuscular injection. After administration, the subject's immunity to S. pneumoniae disease, which is prevalent in Japan and Korea, is improved. Example 14 6C-CRM 197 Methods for Making Pharmaceutical Compositions of Conjugates

[0113] This example demonstrates a method for making a saccharide-polypeptide conjugate pharmaceutical composition. Capsular polysaccharide of serotype 6C is isolated from S. pneumoniae. CRM 197 The 6C polysaccharide is mixed with a CDAP solution and then incubated at a temperature appropriate for polysaccharide activation of the 6C. 197 The calculated amount of 6C-CRM is added to the activated 6C polysaccharide. The reaction is then quenched using glycine or any other suitable quenching reagent. The conjugation reaction mixture is filtered, diafiltered, and concentrated to give 6C-CRM. 197 Then, 6C-CRM 197 The conjugate is purified by gel filtration chromatography. 197 The fractions containing the conjugate are pooled, concentrated, and then filtered through a 0.2 μm filter. 197 The conjugate is formulated for use in a vaccine and contacted with an excipient. Example 15 20B-CRM 197 Methods for Making Pharmaceutical Compositions of Conjugates

[0114] This example demonstrates a method for making a saccharide-polypeptide conjugate pharmaceutical composition. Capsular polysaccharide of serotype 20B is isolated from S. pneumoniae. CRM 197 The 20B polysaccharide is mixed with a CDAP solution and then incubated at a temperature appropriate for polysaccharide activation of the 20B. 197 The calculated amount of 20B polysaccharide is added to the activated 20B polysaccharide. The reaction is then quenched using glycine or any other suitable quenching reagent. The conjugation reaction mixture is filtered, diafiltered, and concentrated to give 20B-CRM. 197 Then, 20B-CRM 197The conjugate is purified by gel filtration chromatography. 197 The fractions containing the conjugate are pooled, concentrated, and then filtered through a 0.2 μm filter. 197 The conjugate is formulated for use in a vaccine and contacted with an excipient. Example 16 Compositions of Kits Containing Saccharide-Polypeptide Conjugates

[0115] This example illustrates the composition of a kit containing a saccharide-polypeptide conjugate. The saccharide-polypeptide conjugate in a vial or pre-filled syringe is placed in a container. The container is further associated with instructions for use. Example 17 Vaccination against S. pneumoniae

[0116] This example demonstrates vaccine compositions and their administration to human subjects. The aqueous vaccine compositions are composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. The aqueous vaccines are formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0117] A single 0.5 mL dose of the aqueous vaccine is given to a human subject by intramuscular injection. After 28 days, blood is drawn from the human subject and tested for antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine by an in vitro opsonization assay. After administration of the vaccine, the human subject is found to have antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine, thus improving the human subject's immunity against these serotypes of S. pneumoniae. Example 18 Vaccination against S. pneumoniae

[0118] This example demonstrates vaccine compositions and their administration to human subjects. The aqueous vaccine compositions are composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. The aqueous vaccines are formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0119] A single 0.5 mL dose of the aqueous vaccine is given to a human subject by intramuscular injection. After 28 days, blood is drawn from the human subject and tested for antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine by an in vitro opsonization assay. After administration of the vaccine, the human subject is found to have antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine, thus improving the human subject's immunity against these serotypes of S. pneumoniae. Example 19 Vaccination against S. pneumoniae

[0120] This example demonstrates vaccine compositions and their administration to human subjects. The aqueous vaccine compositions are composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides derived from any of the S. pneumoniae serotypes in the combinations listed in Table 2. The aqueous vaccines are formulated to contain 1-5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0121] A single 0.5 mL dose of the aqueous vaccine is given to a human subject by intramuscular injection. After 28 days, blood is drawn from the human subject and tested for antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine by an in vitro opsonization assay. After administration of the vaccine, the human subject is found to have antibodies against each serotype of the polysaccharide of the immunogenic saccharide-polypeptide conjugate in the vaccine, thus improving the human subject's immunity against these serotypes of S. pneumoniae. Example 20 Vaccination method

[0122] This example illustrates vaccine compositions and their administration schedules to human subjects. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. Each aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0123] A single 0.5 mL dose of the first aqueous vaccine is given to a human subject via intramuscular injection. Four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given to the human subject via intramuscular injection. Instead of four weeks later, one year later, a single 0.5 mL dose of the second aqueous vaccine is given to the human subject via intramuscular injection. Example 21 Vaccination method

[0124] This example illustrates vaccine compositions and their administration schedules to human subjects. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. Each aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0125] A single 0.5 mL dose of the first aqueous vaccine is given to a human subject via intramuscular injection. Four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given to the human subject via intramuscular injection. Instead of four weeks later, one year later, a single 0.5 mL dose of the second aqueous vaccine is given to the human subject via intramuscular injection. Example 22 Vaccination for adults 50 years of age or older

[0126] This example illustrates vaccine compositions and administration schedules for the vaccine compositions to adults 50 years of age or older. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. Alternatively, the second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. Each aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0127] A single 0.5 mL dose of the first aqueous vaccine is given intramuscularly to adults 50 years of age or older. Four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to adults 50 years of age or older. Instead of four weeks later, one year later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to adults 50 years of age or older. Example 23 Vaccination instructions for children aged 2 to 18 years

[0128] This example illustrates vaccine compositions and administration schedules for the vaccine compositions for children aged 2 to 18 years. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. Alternatively, the second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. Each aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0129] A single 0.5 mL dose of the first aqueous vaccine is given intramuscularly to children aged 2 to 18 years. Four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to children aged 2 to 18 years. One year later, instead of four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to children aged 2 to 18 years. Example 24 Vaccination method for children aged 4 weeks to 2 years

[0130] This example illustrates vaccine compositions and administration schedules for the vaccine compositions for children aged 4 weeks to 2 years. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. Alternatively, the second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae serotypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. Each aqueous vaccine is formulated to contain 1 to 5 micrograms of each serotype and aluminum phosphate as an adjuvant.

[0131] A single 0.5 mL dose of the first aqueous vaccine is given intramuscularly to children 4 weeks to 2 years of age. Four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to children 2 to 18 years of age. One year later, instead of four weeks later, a single 0.5 mL dose of the second aqueous vaccine is given intramuscularly to children 4 weeks to 2 years of age. Example 25 Vaccination strategies against S. pneumoniae and N. meningitidis

[0132] This example demonstrates vaccine compositions and administration schedules that provide protection against S. pneumoniae and N. meningitidis. The first aqueous vaccine composition was CRM 197The second aqueous vaccine composition is comprised of an immunogenic saccharide-polypeptide conjugate containing capsular polysaccharides from S. pneumoniae 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B conjugated to CRM. 197 Each vaccine is formulated for intramuscular injection.

[0133] A single 0.5 mL dose of the first aqueous vaccine is given to the subject via intramuscular injection. One month later, a single dose of the second aqueous vaccine is given to the subject via intramuscular injection. Alternatively, one month after the second aqueous vaccine is given via intramuscular injection, the first aqueous vaccine is given via intramuscular injection. Example 26 Vaccination methods against S. pneumoniae and S. enterica

[0134] This example demonstrates vaccine compositions and administration schedules that provide protection against S. pneumoniae and S. enterica. The first aqueous vaccine composition was CRM 197 The second aqueous vaccine composition is comprised of an immunogenic saccharide-polypeptide conjugate containing capsular polysaccharides from S. pneumoniae 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B conjugated to CRM. 197 Each aqueous vaccine is composed of an immunogenic saccharide-polypeptide conjugate containing the capsular polysaccharide of S. enterica subspecies enterica serovar Typhi Vi conjugated to a saccharide-polypeptide conjugate. Each aqueous vaccine is formulated for intramuscular injection.

[0135] A single 0.5 mL dose of the first aqueous vaccine is administered to the subject via intramuscular injection. One month later, a single dose of the second aqueous vaccine is administered to the subject via intramuscular injection. Alternatively, one month after the second aqueous vaccine is administered via intramuscular injection, the first aqueous vaccine is administered via intramuscular injection. Example 27 Vaccination methods against S. pneumoniae, N. meningitidis, and H. influenzae

[0136] This example demonstrates vaccine compositions and administration schedules that confer protection against S. pneumoniae, N. meningitidis, and H. influenzae. The first aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing capsular polysaccharides from S. pneumoniae subtypes 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B conjugated to a tetanus toxoid carrier protein. The second aqueous vaccine composition is composed of immunogenic saccharide-polypeptide conjugates containing N. meningitidis group C and group Y saccharides and H. influenzae type b saccharides, individually conjugated to a tetanus toxoid carrier protein. Each aqueous vaccine is formulated for intramuscular injection.

[0137] A single 0.5 mL dose of the first aqueous vaccine is administered to the subject via intramuscular injection. One month later, a single dose of the second aqueous vaccine is administered to the subject via intramuscular injection. Alternatively, one month after the second aqueous vaccine is administered via intramuscular injection, the first aqueous vaccine is administered via intramuscular injection.

[0138] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The specific examples provided herein are not intended to limit the present invention. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous modifications, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, shapes, or relative proportions set forth herein, depending upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. It is therefore intended that the present invention also cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. The present invention provides, for example, the following items. (Item 1) 1. A pharmaceutical composition comprising a plurality of at least eight unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38. (Item 2) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least nine unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 35F, and 38. (Item 3) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 10 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35B, 33F, and 38. (Item 4) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 5) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 14 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 6) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptides, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 7) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 12 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38. (Item 8) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 9) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 10) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 11) 10. The pharmaceutical composition of claim 1, comprising a plurality of at least 12 unique immunogenic saccharide-polypeptide conjugates, each comprising a unique serotype selected from the group consisting of 6C, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B and 38. (Item 12) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 11 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 13) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 11 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 14) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 15) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 16 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 20B, 22F, 23A, 23B, 33F, and 35B. (Item 16) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least eight unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B. (Item 17) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least seven unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 15A, 15C, 16F, 23A, 23B, and 33B. (Item 18) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 19) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 20) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. (Item 21) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 9N, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31, 34, 35F, 35B, and 38. (Item 22) 2. The pharmaceutical composition of claim 1, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 6C, 9N, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 24F, 31, 34, 35F, 35B, and 38. (Item 23) 23. The pharmaceutical composition according to any one of items 1 to 22, wherein the specific Streptococcus pneumoniae serotype is not any of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. (Item 24) 1. A pharmaceutical composition comprising a plurality of at least 29 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 34, 35B, and 38. (Item 25) 25. The pharmaceutical composition of claim 24, comprising a plurality of at least 15 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 33F, 35F, 35F, 35B, and 38. (Item 26) 25. The pharmaceutical composition of claim 24, comprising a plurality of at least 25 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 3, 4, 6A, 6C, 7F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 24B, 33F, 35F, 35F, 35B, and 38. (Item 27) 25. The pharmaceutical composition of claim 24, comprising a plurality of at least 27 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 3, 4, 6A, 6B, 6C, 7F, 8, 9N, 10A, 11A, 12F, 14, 15A, 15B, 16F, 18C, 19F, 19A, 20A, 20B, 22F, 23F, 23A, 23B, 24F, 24F, 33F, 35F, and 35B. (Item 28) 25. The pharmaceutical composition of claim 24, comprising a plurality of at least 19 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 3, 4, 6A, 6B, 6C, 7F, 10A, 11A, 14, 15A, 15B, 19F, 19A, 20A, 20B, 22F, 23F, 23A, 24F, 35B, and 38. (Item 29) 1. A pharmaceutical composition comprising at least two immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof, conjugated to a polypeptide, wherein the capsular polysaccharides, fragments thereof, or combinations thereof are derived from distinct Streptococcus pneumoniae serotype groups, one serotype being 6C and at least one additional serotype being selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, and 38. (Item 30) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 1, 3, 4, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24F, 33F, 35F, 35B, and 38. (Item 31) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 32) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 33) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 34) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B, and 38. (Item 35) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. (Item 36) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 10A, 11A, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. (Item 37) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 9N, 15A, 15C, 16F, 23A, 23B, and 33B. (Item 38) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 15A, 15C, 16F, 23A, 23B, and 33B. (Item 39) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 40) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 41) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype is selected from the group consisting of 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. (Item 42) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 43) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 11A, 12F, 15A, 15B, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 44) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 12F, 15A, 22F, 23A, 23B, 24F, 33F, and 35B. (Item 45) 30. The pharmaceutical composition of item 29, wherein the at least one further serotype consists of 10A, 11A, 15A, 15B, 15C, 22F, 23A, 24B, 33F, 35B and 38. (Item 46) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 12F, 15A, 15B, 22F, 23A, 23B, 33F, and 35B. (Item 47) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 3, 4, 7F, 8, 9N, 11A, 12F, 15A, 15B, 15C, 16F, 19A, 22F, 23A, 23B, and 35B. (Item 48) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 49) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 1, 3, 4, 6A, 7F, 8, 9N, 10A, 11A, 12F, 14, 19A, 19F, 22F, 23A, 23B, 23F, 24F, and 33F. (Item 50) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 20B, 23A, 23B, 33F, and 35B. (Item 51) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 3, 4, 6B, 10A, 15A, 15B, 19A, 19F, 22F, 23F, 24F, and 35B. (Item 52) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 15A, 15C, 16F, 23A, 23B, and 33B. (Item 53) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 23A, 23B, 33B. (Item 54) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 23A, 23B, 33B, and 35B. (Item 55) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 17F, 20A, 23A, 23B, 33B, 34, 35F, 35B, 24F, and 31. (Item 56) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 23A, 23B, 33B, 35F, and 35B. (Item 57) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 23A, 23B, and 33B. (Item 58) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 15A, 15C, 16F, 23A, 23B, and 33B. (Item 59) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 60) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 61) 30. The pharmaceutical composition of item 29, wherein the at least one additional serotype consists of 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B. (Item 62) 1. A pharmaceutical composition comprising at least two immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof, conjugated to a polypeptide, wherein the capsular polysaccharides, fragments thereof, or combinations thereof are derived from distinct Streptococcus pneumoniae serotype groups, one serotype being 20B and at least one further serotype being selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 9V, 14, 18C, 19A, 19F, 23F, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 22F, 23A, 23B, 24F, 31, 33F, 34, 35B, and 38. (Item 63) 63. The pharmaceutical composition of item 62, wherein the at least one additional serotype consists of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, 34, and 35B. (Item 64) 63. The pharmaceutical composition of item 62, wherein the at least one additional serotype is selected from the group consisting of 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 22F, 23A, 23B, 33F, and 35B. (Item 65) 63. The pharmaceutical composition of item 62, wherein the at least one additional serotype is selected from the group consisting of 6C, 15A, 15C, 16F, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B. (Item 66) 63. The pharmaceutical composition of item 62, wherein the at least one additional serotype is selected from the group consisting of 6C, 15A, 15C, 16F, 23A, 23B, 24F, 31, 33B, 35F, and 35B. (Item 67) 1. A pharmaceutical composition comprising an immunogenic saccharide-polypeptide conjugate comprising at least two distinct capsular polysaccharides, fragments thereof, or combinations thereof, individually conjugated to a polypeptide, wherein the at least two distinct capsular polysaccharides, fragments thereof, or combinations thereof are selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35F, 35B, and 38. A pharmaceutical composition derived from a serotype of S. pneumoniae. (Item 68) 68. The pharmaceutical composition of item 67, wherein the at least two unique capsular polysaccharides, fragments thereof, or combinations thereof are derived from a serovar of Streptococcus pneumoniae selected from the group consisting of 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 20B, 22F, 23A, 23B, 33F, and 35B. (Item 69) 68. The pharmaceutical composition of item 67, wherein the at least two unique capsular polysaccharides, fragments thereof, or combinations thereof are derived from a serovar of Streptococcus pneumoniae selected from the group consisting of 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 22F, 23A, 23B, 33F, and 35B. (Item 70) 68. The pharmaceutical composition of item 67, further comprising at least one unique immunogenic saccharide-polypeptide conjugate each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, and 38. (Item 71) 71. The pharmaceutical composition of any one of items 1 to 70, wherein at least one unique immunogenic saccharide-polypeptide conjugate comprises said capsular polysaccharide. (Item 72) 72. The pharmaceutical composition of any one of items 1 to 71, wherein at least one unique immunogenic saccharide-polypeptide conjugate comprises a fragment of said capsular polysaccharide. (Item 73) 73. The pharmaceutical composition of claim 72, wherein the fragment of the capsular polysaccharide comprises a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, or a hexasaccharide. (Item 74) 74. The pharmaceutical composition according to any one of items 1 to 73, wherein at least one polypeptide of the plurality is an oligopeptide. (Item 75) At least one polypeptide of the plurality is a CRM 19775. The pharmaceutical composition of any one of items 1 to 74, comprising an inactivated or mutated pneumococcal pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M. catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, PspA, any fragment thereof, or any combination thereof. (Item 76) 76. The pharmaceutical composition according to any one of items 1 to 75, wherein the polypeptide comprises a mixture of polypeptides. (Item 77) 77. The pharmaceutical composition according to any one of items 1 to 76, wherein the toxic activity of at least one polypeptide of the plurality is at least partially mitigated. (Item 78) 78. The pharmaceutical composition according to any one of items 1 to 77, further comprising an adjuvant. (Item 79) 79. The pharmaceutical composition in unit dosage form according to item 78, wherein the adjuvant is an adjuvant containing aluminum cations. (Item 80) 79. The pharmaceutical composition of item 78, wherein the adjuvant is aluminum phosphate, aluminum sulfate, or aluminum hydroxide. (Item 81) 81. The pharmaceutical composition according to any one of items 1 to 80, in the form of an intramuscularly injectable composition, an intradermally injectable composition, a subcutaneously injectable composition or an intranasally administrable composition. (Item 82) 82. The pharmaceutical composition according to any one of the preceding items, further comprising a chelating agent. (Item 83) 83. The pharmaceutical composition of claim 82, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA). (Item 84) 84. The pharmaceutical composition according to any one of items 1 to 83, which is in the form of a suspension. (Item 85) 85. The pharmaceutical composition according to any one of items 1 to 84, in the form of a homogeneous mixture or a heterogeneous mixture. (Item 86) 86. The pharmaceutical composition according to item 85, in the form of a homogeneous mixture. (Item 87) 87. The pharmaceutical composition according to any one of items 1 to 86, which is contained in a pre-filled syringe. (Item 88) 88. The pharmaceutical composition according to any one of the preceding items, further comprising a surfactant, an emulsifier, or a combination thereof. (Item 89) Item 90. The pharmaceutical composition according to Item 88, comprising the surfactant, wherein the surfactant is a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. 90. The pharmaceutical composition of claim 89, comprising the polysorbate, wherein the polysorbate is polysorbate 80. (Item 91) 90. The pharmaceutical composition of claim 89, comprising the polymer glycol, wherein the polymer glycol is polyethylene glycol. (Item 92) 92. The pharmaceutical composition according to any one of items 1 to 91, further comprising a buffering agent. (Item 93) 93. The pharmaceutical composition of item 92, wherein the buffer is succinate buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, potassium phosphate buffer, or any combination thereof. (Item 94) 94. The pharmaceutical composition according to any one of items 1 to 93, further comprising sterile water. (Item 95) 95. The pharmaceutical composition of any one of items 1 to 94, wherein when administered to a plurality of humans, the administration induces at least one of the following side effects in less than about 10% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash. (Item 96) 96. The pharmaceutical composition of any one of items 1 to 95, which when administered intramuscularly to a human induces an immune response lasting from about 1 week to about 30 years. (Item 97) 97. The pharmaceutical composition of any one of items 1 to 96, wherein the unique immunogenic saccharide-polypeptide conjugate elicits an opsonization response. (Item 98) 98. The pharmaceutical composition according to any one of items 1 to 97, wherein the pharmaceutical composition retains at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, wherein the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm. (Item 99) 99. The pharmaceutical composition according to any one of items 1 to 98, which is in unit dosage form. (Item 100) 99. The pharmaceutical composition according to any one of the preceding items, further comprising a preservative. (Item 101) 101. The pharmaceutical composition according to any one of items 1 to 100, further comprising an antifungal compound or a salt thereof. (Item 102) 102. The pharmaceutical composition according to any one of the preceding items, further comprising a salt. (Item 103) 103. The pharmaceutical composition according to any one of the preceding items, wherein the salt is an inorganic salt. (Item 104) 104. The pharmaceutical composition of any one of items 1 to 103, wherein the immunogenic saccharide-polypeptide conjugate is isolated and purified. (Item 105) 105. The pharmaceutical composition according to any one of items 1 to 104, which is a vaccine. (Item 106) 106. The pharmaceutical composition of any one of items 1 to 105, wherein the immunogenic saccharide-polypeptide conjugates are individually, respectively or collectively present in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the composition. (Item 107) 107. The pharmaceutical composition of any one of items 1 to 106, wherein the immunogenic saccharide-polypeptide conjugate is produced using bacteria. (Item 108) 108. The pharmaceutical composition of any one of items 1 to 107, wherein the immunogenic saccharide-polypeptide conjugate is produced using E. coli. (Item 109) 109. The pharmaceutical composition of any one of items 1 to 108, wherein the immunogenic saccharide-polypeptide comprises the capsular polysaccharide at least partially embedded in the polypeptide. (Item 110) 110. The pharmaceutical composition of any one of items 1 to 109, wherein the immunogenic saccharide-polypeptide comprises the capsular polysaccharide chemically cross-linked to the polypeptide. (Item 111) 111. The pharmaceutical composition of any one of items 1 to 110, wherein the immunogenic saccharide-polypeptide comprises the capsular polysaccharide at least partially chemically cross-linked to the polypeptide. (Item 112) 112. A method comprising the step of administering to a subject a first composition, wherein said first composition is a pharmaceutical composition according to any one of items 1 to 111. (Item 113) 114. The method of claim 112, wherein the step of administering the first composition to the subject occurs at least about 4 weeks before or at least about 4 weeks after the administration of a second composition comprising an immunogenic saccharide-polypeptide conjugate comprising a second polypeptide to the subject, wherein the second polypeptide has at least about 70% length identity, at least about 70% sequence identity, or a combination thereof, to the polypeptide of the first composition. 114. The method of claim 113, wherein the second composition comprises an immunogenic saccharide-polypeptide conjugate comprising a capsular polysaccharide or a fragment of a capsular polysaccharide derived from a serotype of Streptococcus pneumoniae conjugated to the second polypeptide. (Item 115) 115. The method of claim 113 or 114, wherein the second composition comprises a plurality of at least 13 unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. (Item 116) 117. The method of any one of claims 113 to 115, wherein the second composition comprises at least one unique immunogenic saccharide-polypeptide conjugate, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, a fragment thereof, or a combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F. 117. The method of any one of items 113 to 116, wherein the second composition comprises at least one unique immunogenic saccharide-polypeptide conjugate, each individually comprising a capsular polysaccharide, a fragment thereof, or a combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, and 38, and wherein the serotype of the first composition and the serotype of the second composition are distinct. (Item 118) 118. The method of claim 117, wherein the immunogenic saccharide-polypeptide conjugate of the second composition comprises a capsular polysaccharide. (Item 119) 119. The method of claim 118, wherein the immunogenic saccharide-polypeptide conjugate of the second composition comprises a fragment of the capsular polysaccharide. (Item 120) 120. The method of claim 119, wherein the fragment of the capsular polysaccharide of the second composition comprises a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, or a hexasaccharide. (Item 121) 121. The method of any one of items 113 to 120, wherein the second polypeptide is an oligopeptide. (Item 122) The second polypeptide is a CRM 197, tetanus toxoid, diphtheria toxoid, cholera toxoid, pertussis toxoid, inactivated or mutated pneumococcal pneumolysin, pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, PspA, any fragment thereof, or any combination thereof. (Item 123) 123. The method of any one of items 113 to 122, wherein the toxic activity of the second polypeptide is at least partially mitigated. (Item 124) 124. The method of any one of items 113 to 123, wherein the second composition further comprises an adjuvant. (Item 125) Item 125. The method of item 124, wherein the adjuvant is an adjuvant containing aluminum cations. (Item 126) Item 126. The method of item 125, wherein the adjuvant is aluminum phosphate, aluminum sulfate, or aluminum hydroxide. (Item 127) 127. The method of any one of items 113 to 126, wherein the second composition is in the form of an intramuscularly injectable composition, an intradermally injectable composition, a subcutaneously injectable composition, or an intranasally injectable composition. (Item 128) 128. The method of any one of items 113 to 127, wherein the second composition is in a unit dosage form. (Item 129) 129. The method of any one of items 113 to 128, wherein the second composition further comprises a chelating agent. (Item 130) Item 129. The method of item 129, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA). (Item 131) 131. The method according to any one of items 113 to 130, wherein the second composition is in the form of a suspension. (Item 132) 132. The method according to any one of items 113 to 131, wherein the second composition is in the form of a homogeneous mixture or a heterogeneous mixture. (Item 133) 133. The method according to any one of items 113 to 132, wherein the second composition is in the form of a homogeneous mixture. (Item 134) 134. The method of any one of items 113 to 133, wherein the second composition is contained in a pre-filled syringe. (Item 135) 135. The method of any one of items 113 to 134, wherein the second composition further comprises a surfactant, an emulsifier, or a combination thereof. (Item 136) Item 136. The method of item 135, wherein the second composition further comprises the surfactant, and the surfactant is a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. (Item 137) Item 137. The method of item 136, wherein the second composition comprises the polysorbate, and the polysorbate is polysorbate 80. (Item 138) Item 138. The method of item 137, wherein the second composition comprises the polymer glycol, and the polymer glycol is polyethylene glycol. (Item 139) 139. The method of any one of items 113 to 138, wherein the second composition further comprises a buffering agent. (Item 140) Item 139. The method of item 139, wherein the buffer is succinate buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, potassium phosphate buffer, or any combination thereof. (Item 141) 141. The method of any one of items 113 to 140, wherein the second composition further comprises sterile water. (Item 142) 142. The method of any one of items 113 to 141, wherein the second composition, when administered to a plurality of humans, induces at least one of the following side effects in less than about 10% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash. (Item 143) 143. The method of any one of items 113 to 142, wherein the second composition, when administered intramuscularly to a human, elicits an immune response that lasts from about 1 week to about 30 years. (Item 144) 144. The method of any one of items 113 to 143, wherein the unique immunogenic saccharide-polypeptide conjugate of the second composition elicits an opsonization response. (Item 145) 145. The method of any one of items 113 to 144, wherein the second composition, when administered intramuscularly, elicits an immune response that lasts from about 1 week to about 30 years. (Item 146) 146. The method of any one of items 113 to 145, wherein the second composition retains at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm. (Item 147) 147. The method of any one of items 113 to 146, wherein the second composition further comprises a preservative. (Item 148) 148. The method of any one of items 113 to 147, wherein the second composition further comprises an antifungal compound or a salt thereof. (Item 149) 149. The method of any one of items 113 to 148, wherein the second composition further comprises a salt. (Item 150) Item 149. The method of item 149, wherein the salt is an inorganic salt. (Item 151) 151. The method of any one of items 113 to 150, wherein the immunogenic saccharide-polypeptide conjugate of the second composition is isolated and purified. (Item 152) 152. The method of any one of items 113 to 151, wherein the second composition is a vaccine. (Item 153) 153. The method of any one of items 113 to 152, which is a method of vaccinating a subject. (Item 154) Item 154. The method of item 153, wherein the subject is a human. (Item 155) 155. The method of any one of items 153 or 154, wherein the subject or human is a subject or human in need thereof. (Item 156) 156. The method of any one of items 113 to 155, wherein the immunogenic saccharide-polypeptide conjugates of the second composition are individually, respectively or collectively present in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the composition. (Item 157) 157. The method of any one of paragraphs 113 to 156, which when performed does not result in more than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% suppression of the immunity conveyed by the first vaccine containing the immunogenic saccharide-polypeptide conjugate as measured by an antibody response to the saccharide of the immunogenic saccharide-polypeptide conjugate of the second composition, wherein the antibody response is measured by measuring an antibody titer. (Item 158) 158. The method of any one of items 113 to 157, wherein the immunogenic saccharide-polypeptide conjugate of the second composition is produced using bacteria. (Item 159) 159. The method of any one of items 113 to 158, wherein the immunogenic saccharide-polypeptide conjugate of the second composition is produced using E. coli. (Item 160) 159. The method of any one of items 113 to 159, wherein the immunogenic saccharide-polypeptide of the second composition comprises the capsular polysaccharide at least partially embedded in the polypeptide. (Item 161) 161. The method of any one of items 113 to 160, wherein the immunogenic saccharide-polypeptide of the second composition comprises the capsular polysaccharide chemically cross-linked to the polypeptide. (Item 162) 162. The method of any one of items 113 to 161, wherein the immunogenic saccharide-polypeptide of the second composition comprises the capsular polysaccharide at least partially chemically cross-linked to the polypeptide. (Item 163) 1. A method comprising administering to a subject a second immunogenic saccharide-polypeptide conjugate composition at least four weeks after administering a first immunogenic saccharide-polypeptide conjugate composition, the second immunogenic saccharide-polypeptide conjugate composition comprises a first polypeptide having at least about 70% length homology, at least about 70% sequence homology, or a combination thereof, to a second polypeptide of the second immunogenic saccharide-polypeptide conjugate composition; the first immunogenic saccharide-polypeptide conjugate composition comprises a first serotype; The method wherein said second immunogenic saccharide-polypeptide conjugate composition comprises a second serotype, and said first serotype and said second serotype are different. (Item 164) 164. The method of claim 163, wherein the first immunogenic saccharide-polypeptide conjugate composition comprises a capsular polysaccharide. (Item 165) 165. The method of claim 164, wherein the first immunogenic saccharide-polypeptide conjugate composition comprises a fragment of the capsular polysaccharide. (Item 166) 166. The method of claim 165, wherein the fragment of the capsular polysaccharide comprises a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, or a hexasaccharide. (Item 167) 167. The method of any one of items 163 to 166, wherein the first polypeptide is an oligopeptide. (Item 168) 168. The method of any one of items 163 to 167, wherein the second polypeptide is an oligopeptide. (Item 169) The first polypeptide is a CRM 197169. The method of any one of paragraphs 163 to 168, comprising an inactivated or mutated pneumococcal pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M. catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, any fragment thereof, or any combination thereof. (Item 170) 169. The method of any one of items 163 to 169, wherein the toxic activity of the first polypeptide is at least partially mitigated. (Item 171) The second polypeptide is a CRM 197 171. The method of any one of items 163 to 170, comprising an inactivated or mutated pneumococcal pneumococcal surface protein A, pneumococcal adhesion protein A, pneumococcal lipoprotein PsaA, C5a peptidase group A or group B streptococcus, non-capsulated H. influenzae P4 protein, non-capsulated H. influenzae P6 protein, M. catarrhalis uspA, keyhole limpet hemocyanin (KLH), OMPC from N. meningitidis, purified tuberculin protein derivative (PPD), protein D from H. influenzae, PspA, any fragment thereof, or any combination thereof. (Item 172) 172. The method of any one of items 163 to 171, wherein the toxic activity of the second polypeptide is at least partially mitigated. (Item 173) 173. The method of any one of items 163 to 172, wherein the first serotype is derived from the same bacterium as the second serotype. (Item 174) 174. The method of any one of items 163 to 173, wherein the first serotype is from a different bacterium than the second serotype. (Item 175) 175. The method of any one of items 163 to 174, wherein the first serotype is from Streptococcus pneumoniae. (Item 176) 175. The method of any one of items 163 to 174, wherein the first serotype is derived from Neisseria meningitidis, Salmonella enterica, or Haemophilus influenzae. (Item 177) 177. The method of any one of items 163 to 176, wherein the second serotype is from Streptococcus pneumoniae. (Item 178) 177. The method of any one of items 163 to 176, wherein the second bacterium is from Neisseria meningitidis, Salmonella enterica, or Haemophilus influenzae. (Item 179) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F from Streptococcus pneumoniae, and the second serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, or 38 from Streptococcus pneumoniae. (Item 180) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F from Streptococcus pneumoniae, and the second serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, or 38 from Streptococcus pneumoniae. (Item 181) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, and 38 from Streptococcus pneumoniae, and the second serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 182) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, and 38 from Streptococcus pneumoniae, and the second serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 183) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second serotype comprises 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae. (Item 184) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae, and the second serotype comprises 6C, 15A, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae. (Item 185) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae and the second serotype comprises 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae. (Item 186) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae and the second serotype comprises 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae. (Item 187) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae and the second serotype comprises 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae. (Item 188) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 6C, 9N, 15A, 15C, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae, and the second serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 189) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 6C, 15A, 16F, 23A, 23B, and 33B from Streptococcus pneumoniae, and the second serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 190) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 6C, 9N, 15A, 15C, 16F, 17F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae, and the second serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 191) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 6C, 15A, 15C, 16F, 20B, 23A, 23B, 24F, 31, 33B, 34, 35F, and 35B from Streptococcus pneumoniae, and the second serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 192) 179. The method of any one of items 163 to 178, wherein the first serotype comprises 6C, 8, 9N, 10A, 11A, 15A, 15B, 15C, 16F, 20A, 22F, 23A, 23B, 33F, and 35B from Streptococcus pneumoniae, and the second serotype comprises 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 193) 179. The method of any one of paragraphs 163 to 178, wherein the first serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F from Streptococcus pneumoniae, and the second serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae. (Item 194) 179. The method of any one of paragraphs 163 to 178, wherein the first serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, or 23F from Streptococcus pneumoniae, and the second serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, 38, and serogroup 20 (including serotypes 20A and 20B) from Streptococcus pneumoniae. (Item 195) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B) from Streptococcus pneumoniae, and the second serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 196) 179. The method of any one of items 163 to 178, wherein the first serotype comprises at least eight of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 22F, 23A, 23B, 24F, 24B, 31, 33F, 35F, 35B, 38, and serogroup 20 (which includes serotypes 20A and 20B) from Streptococcus pneumoniae, and the second serotype comprises at least one of 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F from Streptococcus pneumoniae. (Item 197) 197. The method of any one of items 163 to 196, wherein the first immunogenic saccharide-polypeptide conjugate composition further comprises an adjuvant. (Item 198) Item 198. The method of item 197, wherein the adjuvant is an adjuvant comprising aluminum cations. (Item 199) Item 199. The method of item 198, wherein the adjuvant is aluminum phosphate, aluminum sulfate, or aluminum hydroxide. (Item 200) 200. The method of any one of items 163 to 199, wherein the first immunogenic saccharide-polypeptide conjugate composition is in the form of an intramuscularly injectable composition, an intradermally injectable composition, a subcutaneously injectable composition, or an intranasally injectable composition. (Item 201) 201. The method of any one of items 163 to 200, wherein the first immunogenic saccharide-polypeptide conjugate composition further comprises a chelating agent. (Item 202) 202. The method of claim 201, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA). (Item 203) 203. The method of any one of items 163 to 202, wherein the first immunogenic saccharide-polypeptide conjugate composition is in the form of a suspension. (Item 204) 204. The method of any one of items 163 to 203, wherein the first immunogenic saccharide-polypeptide conjugate composition is in the form of a homogenous mixture or a heterogenous mixture. (Item 205) 205. The method of any one of items 163 to 204, wherein the first immunogenic saccharide-polypeptide conjugate composition is in the form of a homogenous mixture. (Item 206) 206. The method of any one of items 163 to 205, wherein the first immunogenic saccharide-polypeptide conjugate composition is contained in a pre-filled syringe. (Item 207) 207. The method of any one of items 163 to 206, wherein the first immunogenic saccharide-polypeptide conjugate composition further comprises a surfactant or an emulsifier, or a combination thereof. (Item 208) 208. The method of claim 207, wherein the first immunogenic saccharide-polypeptide composition comprises the surfactant, and the surfactant is a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. (Item 209) 209. The method of claim 208, wherein the first immunogenic saccharide-polypeptide composition comprises the polysorbate, and the polysorbate is polysorbate 80. (Item 210) 209. The method of claim 208, wherein the first immunogenic saccharide-polypeptide composition comprises the polymer glycol, and the polymer glycol is polyethylene glycol. (Item 211) 211. The method of any one of items 163 to 210, wherein the first immunogenic saccharide-polypeptide conjugate composition further comprises a buffering agent. (Item 212) Item 212. The method of item 211, wherein the buffer is succinate buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, potassium phosphate buffer, or any combination thereof. (Item 213) 213. The method of any one of items 163 to 212, wherein the first immunogenic saccharide-polypeptide conjugate composition further comprises sterile water. (Item 214) 214. The method of any one of items 163 to 213, wherein the first immunogenic saccharide-polypeptide conjugate composition, when administered to a plurality of humans, induces at least one of the following side effects in less than about 10% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash. (Item 215) 215. The method of any one of paragraphs 163 to 214, wherein the first immunogenic saccharide-polypeptide conjugate composition, when administered intramuscularly to a human, elicits an immune response that lasts from about 1 week to about 30 years. (Item 216) 216. The method of any one of paragraphs 163 to 215, wherein the first immunogenic saccharide-polypeptide conjugate composition elicits an opsonization response. (Item 217) 217. The method of any one of items 163 to 216, wherein the first immunogenic saccharide-polypeptide conjugate composition retains at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm. (Item 218) 218. The method of any one of items 163 to 217, wherein the second immunogenic saccharide-polypeptide conjugate composition further comprises an adjuvant. (Item 219) 219. The method of claim 218, wherein the adjuvant is an adjuvant comprising aluminum cations. (Item 220) 219. The method of claim 218, wherein the adjuvant is aluminum phosphate, aluminum sulfate, or aluminum hydroxide. (Item 221) 221. The method of any one of items 163 to 220, wherein the second immunogenic saccharide-polypeptide conjugate composition is in the form of an intramuscularly injectable composition, an intradermally injectable composition, a subcutaneously injectable composition, or an intranasally injectable composition. (Item 222) 222. The method of any one of items 163 to 221, wherein the second immunogenic saccharide-polypeptide conjugate composition further comprises a chelating agent. (Item 223) 223. The method of claim 222, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA). (Item 224) 224. The method of any one of items 163 to 223, wherein the second immunogenic saccharide-polypeptide conjugate composition is in the form of a suspension. (Item 225) 225. The method of any one of items 163 to 224, wherein the second immunogenic saccharide-polypeptide conjugate composition is in the form of a homogenous mixture or a heterogenous mixture. (Item 226) 226. The method of claim 225, wherein the second immunogenic saccharide-polypeptide conjugate composition is in the form of a homogenous mixture. (Item 227) 227. The method of any one of items 163 to 226, wherein the second immunogenic saccharide-polypeptide conjugate composition is contained in a pre-filled syringe. (Item 228) 228. The method of any one of items 163 to 227, wherein the second immunogenic saccharide-polypeptide conjugate composition further comprises a surfactant, an emulsifier, or a combination thereof. (Item 229) 229. The method of claim 228, wherein the second immunogenic saccharide-polypeptide conjugate composition comprises the surfactant, wherein the surfactant is a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. (Item 230) 230. The method of claim 229, wherein the second immunogenic saccharide-polypeptide conjugate composition comprises the polysorbate, and the polysorbate is polysorbate 80. (Item 231) 231. The method of claim 230, wherein the second immunogenic saccharide-polypeptide conjugate composition comprises the polymer glycol, and the polymer glycol is polyethylene glycol. (Item 232) 232. The method of any one of items 163 to 231, wherein the second immunogenic saccharide-polypeptide conjugate composition further comprises a buffering agent. (Item 233) Item 233. The method of item 232, wherein the buffer is succinate buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, potassium phosphate buffer, or any combination thereof. (Item 234) 234. The method of any one of items 163 to 233, wherein the second immunogenic saccharide-polypeptide conjugate composition further comprises sterile water. (Item 235) 235. The method of any one of items 163 to 234, wherein the second immunogenic saccharide-polypeptide conjugate composition, when administered to a plurality of humans, induces at least one of the following side effects in less than about 10% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash. (Item 236) 236. The method of any one of paragraphs 163 to 235, wherein the second immunogenic saccharide-polypeptide conjugate composition, when administered intramuscularly to a human, elicits an immune response that lasts from about 1 week to about 30 years. (Item 237) 237. The method of any one of paragraphs 163 to 236, wherein the second immunogenic saccharide-polypeptide conjugate composition elicits an opsonization response. (Item 238) 238. The method of any one of paragraphs 163 to 237, wherein the second immunogenic saccharide-polypeptide conjugate composition retains at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm. (Item 239) 239. The method of any one of items 163 to 238, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition is in unit dosage form. (Item 240) 239. The method of any one of items 163 to 239, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition further comprises a preservative. (Item 241) 241. The method of any one of items 163 to 240, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition further comprises an antifungal compound or a salt thereof. (Item 242) 242. The method of any one of items 163 to 241, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition further comprises a salt. (Item 243) 243. The method of claim 242, wherein the salt is an inorganic salt. (Item 244) 244. The method of any one of items 163 to 243, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition is isolated and purified. (Item 245) 245. The method of any one of items 163 to 244, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition is a vaccine. (Item 246) 246. The method of any one of items 163 to 245, which is a method of vaccinating a subject. (Item 247) 247. The method of claim 246, wherein the subject is a human. (Item 248) 248. The method according to item 246 or 247, wherein the subject or human is a subject or human in need thereof. (Item 249) 249. The method of any one of items 163 to 248, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition comprise immunogenic saccharide-polypeptide conjugates present individually, each or collectively in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the composition. (Item 250) 249. The method of any one of paragraphs 163 to 249, wherein administration of the second immunogenic saccharide-polypeptide conjugate composition does not result in more than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% suppression of immunity as measured by an antibody response to the saccharide of the immunogenic saccharide-polypeptide conjugate, wherein the antibody response is measured by measuring an antibody titer. (Item 251) 251. The method of any one of items 163 to 250, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition is produced using bacteria. (Item 252) 252. The method of any one of items 163 to 251, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition is produced using E. coli. (Item 253) 253. The method of any one of items 163 to 252, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition comprises a capsular polysaccharide at least partially embedded in the polypeptide. (Item 254) 254. The method of any one of items 163 to 253, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition comprises a capsular polysaccharide chemically cross-linked to the polypeptide. (Item 255) 255. The method of any one of items 163 to 254, wherein the first immunogenic saccharide-polypeptide conjugate composition and / or the second immunogenic saccharide-polypeptide composition comprises a capsular polysaccharide at least partially chemically cross-linked to the polypeptide. (Item 256) 71. A method of making a composition, comprising the step of contacting the immunogenic saccharide-polypeptide conjugate of any one of items 1 to 70 with an excipient, an adjuvant, or any combination thereof. (Item 257) 257. The method of claim 256, wherein the composition is injectable or intranasally administrable. (Item 258) Item 259. The method of item 256, comprising contacting with an adjuvant, wherein the adjuvant is aluminum phosphate, aluminum sulfate, or aluminum hydroxide. 259. The method of any one of items 256 to 258, wherein the composition is in the form of an intramuscularly injectable composition. (Item 260) 259. The method of any one of items 256 to 259, further comprising contacting the composition with a chelating agent. (Item 261) 261. The method of claim 260, further comprising contacting the composition with a chelating agent, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA). (Item 262) 262. The method according to any one of items 256 to 261, wherein the composition is in the form of a suspension. (Item 263) 263. The method according to any one of items 256 to 262, wherein the composition is in the form of a homogeneous mixture or a heterogeneous mixture. (Item 264) Item 265. The method of any one of items 263, wherein the composition is in the form of a homogeneous mixture. 265. The method of any one of items 256 to 264, wherein the composition is contained in a pre-filled syringe. (Item 266) 266. The method of any one of items 256 to 265, further comprising contacting the composition with a surfactant, an emulsifier, or a combination thereof. (Item 267) 267. The method of claim 266, comprising contacting the composition with a surfactant, wherein the surfactant is a polysorbate, a polymeric glycol, a sorbitan ester, or any combination thereof. (Item 268) 268. The method of claim 267, comprising contacting the composition with the polysorbate, wherein the polysorbate is polysorbate 80. (Item 269) 269. The method of claim 268, comprising contacting the composition with the polymeric glycol, wherein the polymeric glycol is polyethylene glycol. (Item 270) 269. The method of any one of items 256 to 269, further comprising contacting the composition with a buffer. (Item 271) Item 271. The method of item 270, wherein the buffer is succinate buffer, tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, phosphate buffer, sodium phosphate buffer, potassium phosphate buffer, or any combination thereof. (Item 272) 272. The method of any one of items 256 to 271, further comprising contacting the composition with sterile water. (Item 273) 274. The method of any one of claims 256 to 272, wherein the composition, when administered to a plurality of humans, induces at least one of the following side effects in less than about 10% of the plurality of humans: fatigue, headache, muscle pain, joint pain, loss of appetite, chills, or rash. 274. The method of any one of items 256 to 273, wherein the composition, when administered intramuscularly to a human, elicits an immune response that lasts from about 1 week to about 30 years. (Item 275) 275. The method of any one of items 256 to 274, wherein the immunogenic saccharide-polypeptide conjugate elicits an opsonization response. (Item 276) 276. The method of any one of items 256 to 275, wherein the composition retains at least about 80% of its original biological activity when stored in a sealed container at a temperature of about 2°C to 8°C for about 24 months, and the sealed container is stored in an atmosphere having a relative humidity of about 50% and an atmospheric pressure of 1 atm. (Item 277) 277. The method of any one of items 256 to 276, wherein the composition further comprises a preservative. (Item 278) 278. The method of any one of items 256 to 277, wherein the composition further comprises an antifungal compound or a salt thereof. (Item 279) 279. The method of claim 256, wherein the composition further comprises a salt. 279. The method of claim 279, wherein the salt is an inorganic salt. (Item 281) 281. The method of any one of items 256 to 280, wherein the immunogenic saccharide-polypeptide conjugate of the composition is isolated and purified. (Item 282) 282. The method of any one of items 256 to 281, wherein the composition is used as a vaccine. (Item 283) 283. The method of any one of items 256 to 282, wherein the composition is used to vaccinate a subject. (Item 284) 284. The method of claim 283, wherein the subject is a human. (Item 285) 285. The method of any one of items 267 to 284, wherein the subject or human is a subject or human in need thereof. (Item 286) 286. The method of any one of items 256 to 285, wherein the immunogenic saccharide-polypeptide conjugates of the composition are individually, respectively or collectively present in an amount of about 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% by weight of the composition. (Item 287) 287. The method of any one of items 256 to 286, wherein the immunogenic saccharide-polypeptide conjugate of the composition is produced using bacteria. (Item 288) 288. The method of any one of items 256 to 287, wherein the immunogenic saccharide-polypeptide conjugate of the composition is produced using E. coli. (Item 289) 289. The method of any one of items 256 to 288, wherein the immunogenic saccharide-polypeptide conjugate of the composition comprises a capsular polysaccharide at least partially embedded in the polypeptide. (Item 290) 289. The method of any one of items 256 to 289, wherein the immunogenic saccharide-polypeptide conjugate of the composition comprises a capsular polysaccharide chemically cross-linked to the polypeptide. (Item 291) 291. The method of any one of items 256 to 290, wherein the immunogenic saccharide-polypeptide conjugate of the composition comprises a capsular polysaccharide at least partially chemically cross-linked to the polypeptide. (Item 292) 112. A kit comprising the pharmaceutical composition of any one of items 1 to 111 contained in a container. (Item 293) 293. The kit of item 292, further comprising instructions for use. (Item 294) 294. The kit of item 292 or 293, wherein the container is a syringe. (Item 295) 295. The kit of any one of items 292 to 294, wherein the container is a vial including a cap, the cap including a rubber stopper.

Claims

[Claim 1] A pharmaceutical composition comprising a plurality of at least eight unique immunogenic saccharide-polypeptide conjugates, each individually comprising a capsular polysaccharide, fragment thereof, or combination thereof conjugated to a polypeptide, wherein the capsular polysaccharide, fragment thereof, or combination thereof is derived from a unique Streptococcus pneumoniae serotype selected from the group consisting of 2, 6C, 8, 9N, 10A, 11A, 12F, 15A, 15B, 15C, 16F, 17F, 20A, 20B, 22F, 23A, 23B, 24F, 24B, 31, 33F, 34, 35B, 35F, and 38.

Images