Improved methods for producing bacterial capsular saccharide glycoconjugates

Abstract

The disclosure provides, among other embodiments, improved bacterial capsular saccharide glycoconjugates, improved methods of producing such glycoconjugates, immunogenic compositions comprising such glycoconjugates, and uses thereof.

Description

[0001]PC072948A IMPROVED METHODS FOR PRODUCING BACTERIAL CAPSULAR SACCHARIDEGLYCOCONJUGATES BACKGROUND Certain bacteria of medical and public health importance produce an extracellular capsule, principally composed of polysaccharides, which surrounds the bacterial cells and can prevent antibody recognition of antigens in the cell wall. Often relatively poorly immunogenic on their own, the antigenic potential of bacterial capsule polysaccharides can be improved by conjugating them to different so-called carrier proteins, forming glycoconjugates, which have demonstrated their effectiveness in vaccines against bacterial pathogens, such as Streptococcus pneumoniae. Existing methods for producing glycoconjugates involve separately reacting carrier proteins andcapsular polysaccharides with compounds to create reactive sites on each, and then reacting the activated carrier and polysaccharides to cross-link, or conjugate them. The degree of activation can be hard to control, however, so that the relative amount of polysaccharide which ends up conjugated to carrier canvary significantly from one preparation to another. To produce multivalent vaccines, this limitation canbe compensated for by mixing different amounts of glycoconjugates, each with a single type ofpolysaccharide conjugated to carrier. This strategy, however, is not well suited to making multivalentvaccines in which glycoconjugates contain at least two types of polysaccharide simultaneously conjugatedto carrier which would be a more efficient way of producing such vaccines.Accordingly, there exists a need in the art for methods of conjugating multiple types of bacterial capsular polysaccharides to carrier protein at the same time, where the relative amounts of thepolysaccharides in the glycoconjugates, both to each other and to the carrier, are more controllable andpredictable. There also exists a need in the art for glycoconjugates which could be produced by suchmethods so that multivalent vaccines can be produced more efficiently and reliably.SUMMARY Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following embodiments (E).E1. A method of producing a glycoconjugate comprising reacting an alkyne functionalized carrierprotein with a first and at least a second azido activated capsular saccharide of distinct bacterial serotypes in the same azide-alkyne cycloaddition reaction using Cu(I) as a catalyst.E2. The method of embodiment 1, wherein the first and at least second azido activated saccharidesare added to the reaction in a mass ratio ranging from about 1:1 to about 1:5.E3. The method of embodiment 2, wherein the first and at least second azido activated saccharidesare added to the reaction in a mass ratio of about 1:2.E4. The method of embodiment 2, wherein the first and at least second azido activated saccharidesare added to the reaction in a mass ratio of about 1:1.E5. The method of embodiment 4, wherein the relative masses of the first and at least second azidoactivated saccharides differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less.E6. The method of any one of embodiments 1 to 5, wherein the reaction comprises azido activatedcapsular saccharides of at least 3 distinct bacterial serotypes.E7. The method of any one of embodiments 1 to 5, wherein the reaction comprises azido activatedcapsular saccharides of at least 4 distinct bacterial serotypes.E8. The method of any one of embodiments 1 to 5, wherein the reaction comprises azido activatedcapsular saccharides of at least 5 distinct bacterial serotypes.E9. The method of any one of embodiments 1 to 8, wherein said alkyne functionalized carrier proteinis prepared by reacting a carrier protein with a compound comprising an N-hydroxysuccinimide (NHS)moiety and an alkyne group, wherein said NHS moiety reacts with an amino group of said carrier protein,forming an amide linkage.E10. The method of embodiment 9, wherein the alkyne of said compound is a terminal alkyne or acycloalkyne.E11. The method of any one of embodiments 9 to 10, wherein said compound has the general formula(I): where X is selected from the group consisting of CH2O(CH2)nCH2C=O and CH2O(CH2CH2O)m(CH2)nCH2C=O, n ranges from 0 to 10, and m ranges from 0 to 4.E12. The method of any one of embodiments 9 to 11, wherein said compound has the formula (II): E13. The method of any one of embodiments 9 to 12, wherein said carrier protein is reacted with anamount of said compound that is between 0.10 to 10.00 molar equivalents to the amount of lysine in said carrier protein.E14. The method of any one of embodiments 1 to 13, wherein the degree of functionalization of saidalkyne functionalized carrier protein ranges from 1 to 50.E15. The method of any one of embodiments 1 to 8, wherein the azido activated capsular saccharidesare prepared by reacting a capsular saccharide with a carbonic acid derivative and an azido linker.E16. The method of embodiment 15, wherein said azido linker has the formula (III):(III), where X is selected from the group consisting of CH2(CH2)n, (CH2CH2O)mCH2CH2, NHCO(CH2)n, NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n, and O(CH2CH2O)mCH2CH2, n ranges from 1 to 10, and m ranges from 1 to 4.E17. The method of any one of embodiments 15 to 16, wherein said azido linker has the formula (IV): (IV).E18. The method of embodiment 15, wherein either or both of said steps of reacting with thecarbonic acid derivative or azido linker are carried out in an aprotic solvent.E19. The method of any one of embodiments 15 to 18, wherein said step of reacting with thecarbonic acid derivative is carried out in a solution comprising dimethylsulphoxide (DMSO).E20. The method of any one of embodiments 15 to 19, wherein the carbonic acid derivative is selectedfrom the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.E21. The method of embodiment 18, wherein the carbonic acid derivative is CDI and the aproticsolvent comprises 0.1% to 1% (v / v) water.E22. The method of embodiment 19, wherein the carbonic acid derivative is CDI and the solutioncomprising DMSO comprises 0.1% to 1% (v / v) water.E23. The method of embodiments 15, further comprising, after reacting the capsular saccharide withthe carbonic acid derivative, adding water.E24. The method of embodiment 23, wherein the total water content of the reaction ranges fromabout 1% to about 10% (v / v).E25. The method of any one of embodiments 15 to 24, wherein the reaction with the azido linker iscarried out with an amount of azido linker that is between 0.01 to 10.00 molar equivalents of the amount of saccharide repeat units in the capsular saccharides.E26. The method of any one of embodiments 15 to 25, wherein the degree of activation of the azidoactivated capsular saccharides ranges from 0.5% to 50%.E27. The method of any one of embodiments 1 to 26, wherein the weight average molecular weight(Mw) of the capsular saccharides ranges from 10 kDa to 2000 kDa.E28. The method of any one of embodiments 1 to 27, wherein the reaction further comprises areducing agent.E29. The method of any one of embodiments 1 to 28, wherein the reaction further comprises tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and aminoguanidine, wherein reducing agent is ascorbate.E30. The method of any one of embodiments 1 to 29, wherein the reaction comprises azido activatedcapsular saccharides and alkyne functionalized carrier protein in a mass ratio ranging from 0.1 to 3.0 (w / w).E31. The method of any one of embodiments 1 to 30, further comprising capping unreacted azidogroups with an azido group capping agent.E32. The method of embodiment 31, wherein the azido group capping agent is a compound offormula (V), (V), where X is (CH2)n and where n is selected from 1 to 15.E33. The method of embodiment 31, wherein the azido group capping agent is propargyl alcohol.E34. The method of any one of embodiments 31 to 33, wherein capping azido groups is carried outwith an amount of the capping agent that is between 0.05 to 20.00 molar equivalents to the amount of saccharide repeat units of the capsular saccharide.E35. The method of any one of embodiments 1 to 30, further comprising capping unreacted alkynegroups with an alkyne group capping agent.E36. The method of embodiment 35, wherein the alkyne group capping agent comprises an azidogroup.E37. The method of embodiment 35, wherein the alkyne group capping agent is a compound offormula (VI), (VI), where X is (CH2)n and where n is selected from 1 to 15.E38. The method of embodiment 35, wherein the alkyne group capping agent is 3-azido-1-propanol.E39. The method of any one of embodiments 32-35, wherein capping alkyne groups is carried out withan amount of the capping agent that is between 0.05 to 20.00 molar equivalents to the amount of saccharide repeat units of said capsular saccharide or to the amount of lysine in said carrier protein.E40. The method of any one of embodiments 1 to 39, further comprising purifying the glycoconjugate.E41. The method of any one of embodiments 1 to 40, wherein the carrier protein is selected from thegroup consisting of: CRM197; diphtheria toxoid (DT); tetanus toxoid (TT); H. influenzae protein D (PD); SCPor an enzymatically inactive fragment thereof; an enzymatically inactive fragment of an SCP comprising at least one substitution mutation D130A, H193A, N295A or S512A; an enzymatically inactive fragment of an SCP comprising at least the substitution mutations D130A and S512A; an enzymatically inactive fragment of SCP comprising the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence at least 95% identical thereto; and an enzymatically inactive fragment of SCP comprising the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence at least 95% identical thereto.E42. The method of any one of embodiments 1 to 41, wherein the capsular saccharide is from apathogenic bacteria selected from the group consisting of: Streptococcus, Staphylococcus, Enterococcus,Bacillus, Corynebacterium, Listeria, Erysipelothrix, Clostridium, Haemophilus, Neisseria, and Escherichia.E43. The method of any one of embodiments 1 to 41, wherein the capsular saccharide is from apathogenic Streptococcus bacteria selected from the group consisting of: Group B streptococcus (GBS),Group C streptococcus, and Group G streptococcus.E44. The method of any one of embodiments 1 to 41, wherein the capsular saccharide is from apathogenic Group B streptococcus (GBS), bacteria selected from the group consisting of: GBS type Ia, Ib, II, III, IV, V, VI, VII, and VIII.E45. The method of any one of embodiments 1 to 41, wherein the capsular saccharide is from apathogenic bacteria selected from the group consisting of: Enterococcus faecalis, Escherichia coli,Haemophilus influenzae, Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus agalactiae,Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes.E46. The method of any one of embodiments 1 to 41, wherein at least one of the capsular saccharidesis a capsular saccharide from a Streptococcus pneumoniae serotype selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73.E47. A bacterial capsular saccharide glycoconjugate produced by the method of any one ofembodiments 1 to 46.E48. The glycoconjugate of embodiment 47, wherein each of the first and and at second capsularsaccharides is covalently conjugated to said carrier protein (CP) through a spacer and has the general formula (VII): where X is selected from the group consisting of CH2(CH2)n’, (CH2CH2O)mCH2CH2, NHCO(CH2)n’, NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n’ and O(CH2CH2O)mCH2CH2, n' ranges from 1 to 10, and m ranges from 1 to 4; and where X' is selected from the group consisting of CH2O(CH2)n’’CH2C=O, CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, n'' ranges from 0 to 10, and m' ranges from 0 to 4.E49. The glycoconjugate of embodiment 48, where X is CH2(CH2)n’ and n' is 2; and where X' isCH2O(CH2)n’’CH2C=O and n'' is 1.E50. The glycoconjugate of embodiments 47, wherein each of the first and and at second capsularsaccharides is covalently conjugated to the carrier protein (CP) through a spacer and has the general formula (VIII): (VIII).E51. The glycoconjugate of any one of embodiments 47 to 50, wherein the weight average molecularweight (Mw) of either of the first or at least second capsular saccharides before conjugation ranges from 10 kDa to 2,000 kDa.E52. The glycoconjugate of any one of embodiments 47 to 51, wherein the weight average molecularweight (Mw) of the glycoconjugate ranges from 250 kDa to 20,000 kDa.E53. The glycoconjugate of any one of embodiments 47 to 52, wherein the degree of conjugation ofthe glycoconjugate ranges from 2 to 15.E54. The glycoconjugate of any one of embodiments 47 to 53, wherein the mass ratio of total capsularsaccharides to carrier protein in the glycoconjugate ranges from 0.5 to 3.0 on a weight by weight (w / w)basis.E55. The glycoconjugate of any one of embodiments 47 to 54, wherein the glycoconjugate comprisesat least one covalent linkage between the carrier protein and the capsular saccharides for every 4 to 25 saccharide repeat units.E56. The glycoconjugate of any one of embodiments 47 to 55, wherein each of the first and at leastsecond capsular saccharides is a capsular saccharide from distinct Streptococcus pneumoniae (S.pneumoniae) serotypes selected from the group of serotypes consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B,23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73.E57. The glycoconjugate of any one of embodiments 47 to 56, wherein the carrier protein is CRM197.E58. The glycoconjugate of any one of embodiments 47 to 56, wherein the carrier protein is SCP.E59. The glycoconjugate of any one of embodiments 56 to 58, wherein the mass ratio of the first andat least second S. pneumoniae saccharide serotypes conjugated to the carrier protein ranges from about1:1 to about 1:5.E60. The glycoconjugate of any one of embodiments 56 to 58, wherein the mass ratio of the first andat least second S. pneumoniae saccharide serotypes conjugated to the carrier protein is about 1:2.E61. The glycoconjugate of any one of embodiments 56 to 58, wherein the mass ratio of the first andat least second S. pneumoniae saccharide serotypes conjugated to the carrier protein is about 1:1.E62. The glycoconjugate of embodiment 61, wherein the mass ratio of the first and at least second S.pneumoniae saccharide serotypes conjugated to the carrier protein differ from each other by not morethan about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less.E63. The glycoconjugate of any one of embodiments 56 to 62, wherein the glycoconjugate comprisesat least 3 distinct S. pneumoniae saccharide serotypes.E64. The glycoconjugate of any one of embodiments 56 to 62, wherein the glycoconjugate comprisesat least 4 distinct S. pneumoniae saccharide serotypes.E65. The glycoconjugate of any one of embodiments 56 to 62, wherein the glycoconjugate comprisesat least 5 distinct S. pneumoniae saccharide serotypes.E66. An immunogenic composition comprising the glycoconjugate of any one of embodiments 56 to65.E67. The immunogenic composition of embodiment 66, comprising a first and at least secondglycoconjugate, wherein the first glycoconjugate comprises a carrier protein conjugated to at least 2distinct S. pneumoniae saccharide serotypes and the second glycoconjugate comprises a carrier proteinconjugated to at least one S. pneumoniae saccharide serotype which is different from the saccharideserotypes of the first glycoconjugate.E68. The immunogenic composition of embodiment 67, wherein the first glycoconjugate comprises acarrier protein conjugated to at least 3 distinct S. pneumoniae saccharide serotypes.E69. The immunogenic composition of embodiment 67, wherein the first glycoconjugate comprises acarrier protein conjugated to at least 4 distinct S. pneumoniae saccharide serotypes.E70. The immunogenic composition of embodiment 67, wherein the first glycoconjugate comprises acarrier protein conjugated to at least 5 distinct S. pneumoniae saccharide serotypes.E71. A method of preventing a disease or disorder in a subject caused by a bacterial infection,comprising administering a prophylactically effective amount of an immunogenic composition comprising the glycoconjugate of any one of embodiments 56 to 65 or the immunogenic composition of any one of embodiments 66 to 70.E72. A method of treating a disease or disorder in a subject caused by a bacterial infection, comprisingadministering a therapeutically effective amount of an immunogenic composition comprising the glycoconjugate of any one of embodiments 56 to 65 or the immunogenic composition of any one of embodiments 66 to 70.E73. The method of any one of embodiments 71 or 72, wherein the bacterial infection is ofStreptococcus pneumoniae bacteria.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows a general scheme for the preparation of glycoconjugate prepared using click chemistry andusing 3-azido-1-propylamine as azido linker. CP = Carrier Protein, CDI = 1,1'-carbonyldiimidazole.FIG. 2 shows a general scheme for the preparation of glycoconjugate comprising Streptococcuspneumoniae capsular polysaccharides of two different serotypes simultaneously conjugated to carrierprotein.FIG. 3 shows a general scheme for the preparation of glycoconjugate comprising S. pneumoniae capsularpolysaccharides of three different serotypes simultaneously conjugated to carrier protein.FIG. 4 shows results of experiments investigating the immune response of mice after inoculation withglycoconjugates comprising S. pneumoniae capsular polysaccharides prepared by conventional methodsand by click chemistry.FIG. 5 shows results of experiments investigating the immune response of mice after inoculation withglycoconjugates comprising S. pneumoniae capsular polysaccharides prepared by conventional methodsand by click chemistry.FIG. 6 shows results of experiments investigating the immune response of mice after inoculation withglycoconjugates comprising S. pneumoniae capsular polysaccharides prepared by conventional methodsand by click chemistry. DETAILED DESCRIPTION The inventors have surprisingly discovered novel and inventive chemical reactions for synthesizingglycoconjugates comprising a plurality of distinct types of bacterial capsular saccharide antigenscovalently linked to a carrier protein. Compared with previous methods, those described here confersurprisingly greater control over the relative amounts of the different types of saccharides which are conjugated to the carrier. This advance provides more reliable and efficient methods of preparing immunogeniccompositions, including vaccines, having desired molar and / or mass ratios of the different antigenicsaccharide components, a property which is important for the simultaneous efficacy of such immunogeniccompositions against different strains of pathogenic bacteria. In addition to improved methods for theirsynthesis, the present disclosure further provides, among other embodiments, novel glycoconjugatescomprising bacterial capsular saccharide antigens covalently linked to a carrier protein, immunogeniccompositions comprising such glycoconjugates, and methods of treating or preventing diseases ordisorders caused by a bacterial infection by administering such immunogenic compositions to subjects.Bacterial capsular saccharides useful for preparing glycoconjugatesThe term "saccharide" throughout this specification may indicate polysaccharide or oligosaccharide and includes both. In some embodiments, saccharide of the disclosure may be oligosaccharides. Oligosaccharides can have a low number of repeat units (typically 5-15 repeat units) andare typically derived synthetically or by hydrolysis of polysaccharides. In other embodiments though, allof the saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides are able to induce certain antibody immune responses due to the epitopes present on the antigenic surface. The isolation and purification of high molecular weight capsular polysaccharides is contemplated for use in the conjugates, compositions andmethods of the present invention. Therefore, in some embodiments of the present invention, thesaccharide is a polysaccharide. In some embodiments, the saccharide used in the present invention is a bacterial capsularsaccharide (also named “capsular saccharide” herein). Capsules are found in several bacteria of medicalimportance. Bacterial capsules are largely composed of polysaccharides. Capsular saccharides are prepared by standard techniques known to those of ordinary skill in the art. In some embodiments of the present invention, the saccharide is a S. pneumoniae capsularpolysaccharide. In some embodiments, the capsular saccharide used in the present invention is a synthetic carbohydrate. In some embodiments though, the source of bacterial capsular saccharide according to thisinvention can be bacterial cells. Bacterial strains which can be used as source of capsular saccharide may be obtained from established culture collections (such as for example from the American Type CultureCollection (ATCC, Manassas, VA USA) or the Streptococcal Reference Laboratory (Centers for DiseaseControl and Prevention, Atlanta, GA USA)) or clinical specimens. Bacterial capsular saccharides can be obtained directly from bacteria using isolation proceduresknown to one of ordinary skill in the art (see for example methods disclosed in US2006 / 0228380, US2006 / 0228381, US2007 / 0184071, US2007 / 0184072, US2007 / 0231340, and US2008 / 0102498 and WO2008 / 118752). They can also be produced using synthetic protocols known to the man skilled in the art. In case the bacterial capsular saccharide is obtained directly from bacteria, the bacterial cells canbe grown in a medium. Following fermentation of bacterial cells that produce the capsular saccharide, thebacterial cells can be lysed to produce a cell lysate. The capsular saccharide may then be isolated from the cell lysate using purification techniques known in the art, including the use of centrifugation, depth filtration, precipitation, ultra-filtration, treatment with activate carbon, diafiltration and / or column chromatography (see, for example, US2006 / 0228380, US2006 / 0228381 and WO2008 / 118752). The purified capsular saccharide can then be used for the preparation of immunogenic conjugates. The isolated capsular saccharide obtained by purification can be characterized by different parameters including, for example the weight average molecular weight (Mw). The molecular weight of the polysaccharide can be measured by Size Exclusion Chromatography (SEC) combined with Multiangle Laser Light Scattering detector (MALLS). In some embodiments, the capsular saccharide used in the method of making or part of theglycoconjugate of the present invention is a capsular saccharide from a pathogenic bacteria. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide from apathogenic Streptococcus, a pathogenic Staphylococcus, a pathogenic Enterococcus, a pathogenic Bacillus,a pathogenic Corynebacterium, a pathogenic Listeria, a pathogenic Erysipelothrix, a pathogenicClostridium, a pathogenic Haemophilus, a pathogenic Neisseria or a pathogenic Escherichia. In otherembodiments, the capsular saccharide used in the present invention is a capsular saccharide from apathogenic Streptococcus, a pathogenic Neisseria or a pathogenic Escherichia.In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Aeromonas hydrophila and other species (spp.); Bacillus anthracis; Bacillus cereus;Botulinum neurotoxin producing species of Clostridium; Brucella abortus; Brucella melitensis; Brucellasuis; Burkholderia mallei (formally Pseudomonas mallei); Burkholderia pseudomallei (formerlyPseudomonas pseudomallei); Campylobacter jejuni; Chlamydia psittaci; Chlamydia trachomatis, Clostridium botulinum; Clostridium dificile; Clostridium perfringens; Coccidioides immitis; Coccidioidesposadasii; Cowdria ruminantium (Heartwater); Coxiella burnetii; Enterococcus faecalis; EnterovirulentEscherichia coli group (EEC Group) such as Escherichia coli - enterotoxigenic (ETEC), Escherichia coli -enteropathogenic (EPEC), Escherichia coli - O157:H7 enterohemorrhagic (EHEC), and Escherichia coli -enteroinvasive (EIEC); Ehrlichia spp. such as Ehrlichia chajfeensis; Francisella tularensis; Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus; Listeria monocytogenes; miscellaneous enterics such as Klebsiella, Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, and Serratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasma capricolum; Mycoplasma mycoides ssp mycoides; Peronosclerosporaphilippinensis; Phakopsora pachyrhizi; Plesiomonas shigelloides; Ralstonia solanacearum race 3, biovar 2; Rickettsia prowazekii; Rickettsia rickettsii; Salmonella spp.; Schlerophthora rayssiae var zeae; Shigella spp.; Staphylococcus aureus; Streptococcus; Synchytrium endobioticum; Vibrio cholerae non-01; Vibrio cholerae 01; Vibrio par ahaemo Iy ticus and other Vibrios; Vibrio vulnificus; Xanthomonas oryzae; Xylella fastidiosa (citrus variegated chlorosis strain); Yersinia enterocolitica andYersinia pseudotuberculosis; or Yersinia pestis. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Enterococcus faecalis, Escherichia coli, Staphylococcusaureus or Streptococcus.In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from Haemophilus influenzae, Neisseria meningitidis, S. pneumoniae, S. pyogenes, S.agalactiae, Group C & G Streptococci or Escherichia coli. In other embodiments, the capsular saccharideused in the present invention is a capsular saccharide from Neisseria meningitidis, S. pneumoniae, S.agalactiae or Escherichia coli. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from S. pneumoniae or S. agalactiae. In some embodiments, thecapsular saccharide used in the present invention is a capsular saccharide from S. pneumoniae. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Staphylococcus aureus. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Staphylococcus aureus type 5 or Staphylococcus aureus type 8. In some embodiments, the capsular saccharide used in the present invention is is a capsular saccharide from Enterococcus faecalis. In yet a further embodiment, the capsular saccharide used in thepresent invention is a capsular saccharide from is Haemophilus influenzae type b.In a further embodiment, the capsular saccharide used in the present invention is a capsularsaccharide from Neisseria meningitidis. In some embodiments the capsular saccharide used in the presentinvention is a capsular saccharide from N. meningitidis serogroup A (MenA), N. meningitidis serogroupW135 (MenW135), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup X (MenX) or N.meningitidis serogroup C (MenC). In some embodiments the capsular saccharide used in the presentinvention is a capsular saccharide from N. meningitidis serogroup A (MenA). In some embodiments thecapsular saccharide used in the present invention is a capsular saccharide from N. meningitidis serogroupW135 (MenW135). In some embodiments the capsular saccharide used in the present invention is acapsular saccharide from N. meningitidis serogroup Y (MenY). In some embodiments the capsularsaccharide used in the present invention is a capsular saccharide from N. meningitidis serogroup C (MenC).In some embodiments the capsular saccharide used in the present invention is a capsular saccharide fromN. meningitidis serogroup X (MenX).In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Escherichia coli. In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Enterococcus faecalis. In a further embodiment, the capsular saccharide used in the present invention is a capsularsaccharide from Streptococcus agalactiae (Group B streptococcus (GBS)). In some embodiments, thecapsular saccharide used in the present invention is a capsular saccharide from GBS type Ia, Ib, II, III, IV,V, VI, VII or VIII. In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from GBS types Ia, Ib, II, III or V.In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Escherichia coli. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from an Escherichia coli part of the Enterovirulent Escherichia coli group(EEC Group) such as Escherichia coli - enterotoxigenic (ETEC), Escherichia coli - enteropathogenic (EPEC),Escherichia coli - O157:H7 enterohemorrhagic (EHEC), or Escherichia coli - enteroinvasive (EIEC). In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide from anUropathogenic Escherichia coli (UPEC).In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from an Escherichia coli serotype selected from the group consisting of serotypes O157:H7,O26:H11, O111:H- and O103:H2. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting ofserotypes O6:K2:H1 and O18:K1:H7. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting ofserotypes O45:K1, O17:K52:H18, O19:H34 and O7:K1. In some embodiments, the capsular saccharideused in the present invention is a capsular saccharide from an Escherichia coli serotype O104:H4. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide from anEscherichia coli serotype O1:K12:H7. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from an Escherichia coli serotype O127:H6. In some embodiments, thecapsular saccharide used in the present invention is a capsular saccharide from an Escherichia coliserotype O139:H28. In some embodiments, the capsular saccharide used in the present invention is acapsular saccharide from an Escherichia coli serotype O128:H2.In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from Steptococcus pneumoniae. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from a Streptococcus pneumoniae serotype selected from thegroup consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B,15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, glycoconjugates of the disclosurecomprise carrier protein to which are simultaneously conjugated two or more distinct capsularsaccharide serotypes from Steptococcus pneumoniae.In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from Streptococcus pneumoniae serotype 1. In some embodiments, the capsular saccharideused in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 2. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 3. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from Streptococcus pneumoniae serotype 4. In some embodiments, thecapsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 5. In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from Streptococcus pneumoniae serotype 6A. In some embodiments, the capsular saccharideused in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 6B. Insome embodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 7C. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 7F. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 8. In some embodiments, the capsular saccharide used in the presentinvention is a capsular saccharide from Streptococcus pneumoniae serotype 9V. In some embodiments,the capsular saccharide used in the present invention is a capsular saccharide from Streptococcuspneumoniae serotype 9N. In some embodiments, the capsular saccharide used in the present inventionis a capsular saccharide from Streptococcus pneumoniae serotype 10A. In some embodiments, thecapsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 10B. In some embodiments, the capsular saccharide used in the present invention is a capsularsaccharide from Streptococcus pneumoniae serotype 11A. In some embodiments, the capsular saccharideused in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 12F. Insome embodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 14. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 15A. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 15B. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 15C. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 16F. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 17F. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 18C. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 19A. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 19F. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 20. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 20B. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 21. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 22A. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 22F. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 23A. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 23B. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 23F. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 24B. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 24F. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 27. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 29. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 31. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 33B. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 33F. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 34. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 35B. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 35F. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 38. In someembodiments, the capsular saccharide used in the present invention is a capsular saccharide fromStreptococcus pneumoniae serotype 72. In some embodiments, the capsular saccharide used in thepresent invention is a capsular saccharide from Streptococcus pneumoniae serotype 73. Carrier proteins useful for preparing glycoconjugates Capsular saccharide glycoconjugates of the disclosure comprise a carrier protein to which capsular saccharide is conjugated. Any type of carrier protein to which capsular saccharide may be conjugated according to the methods disclosed herein may be used. Alternative terms for carrier protein which may be used herein include “protein carrier” and “carrier”. In some embodiments, the carrier protein of the glycoconjugate is selected in the group consisting of: diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, as well as antigenic nontoxic variants of diphtheria toxin, including CRM197, and others, such as CRM176, CRM228, and CRM45(Uchida et al. (1973) J. Biol. Chem. 218:3838-3844); CRM9, CRM102, CRM103 or CRM107; and other variants described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. (1992); deletion or mutation of Glu-148 to Asp, Gln or Ser and / or Ala 158 to GIy and other mutations disclosed in U.S. Patent Nos. 4,709,017 and 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in U.S. Patent Nos.5,917,017 and 6,455,673; or fragment disclosed in U.S. Patent No. 5,843,711, pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect lmmun 63:2706- 2713) including ply detoxified in some fashion, for example dPLY-GMBS (WO 2004 / 081515, WO 2006 / 032499) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (sequences of PhtA, PhtB, PhtD or PhtE are disclosed in WO 00 / 37105 and WO 00 / 39299) and fusions of Pht proteins, for example PhtDE fusions, PhtBE fusions, Pht A-E (WO 01 / 98334, WO 03 / 054007, WO 2009 / 000826), OMPC (meningococcal outer membrane protein), which is usually extracted from Neisseria meningitidis serogroup B(EP0372501), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D; see, e.g., EP0594610 B),or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (Falugi et al. (2001) Eur J Immunol 31:3816- 3824) such as N19 protein (Baraldoi et al. (2004) Infect lmmun 72:4884-4887) pneumococcal surface protein PspA (WO 02 / 091998), iron uptake proteins (WO 01 / 72337), toxin A or B of Clostridium difficile (WO 00 / 61761), transferrin binding proteins, pneumococcal adhesion protein (PsaA), recombinantPseudomonas aeruginosa exotoxin A (in particular non-toxic mutants thereof (such as exotoxin A bearinga substution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol.169(11):4967-4971)). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) also can be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as cholera toxoid (e.g., as described in WO 2004 / 083251), Escherichiacoli LT, E. coli ST, and exotoxin A from P. aeruginosa. Another suitable carrier protein is a C5a peptidasefrom Streptococcus (SCP). In some embodiments, the carrier protein of the glycoconjugate is CRM197. The CRM197protein is a nontoxic form of diphtheria toxin but is reportedly immunologically indistinguishable from thediphtheria toxin. CRM197 can be produced by Corynebacterium diphtheriae infected by the nontoxigenicphage β197tox-created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al. (1971) Nature New Biology 233:8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin. The CRM197protein is a safe and effective T-cell dependent carrier for saccharides. Further details about CRM197and production thereof can be found, e.g., in U.S. Patent No.5,614,382. In some embodiments, the carrier protein of the glycoconjugate is the A chain of CRM197 (see, e.g., CN103495161). In some embodiments, the carrier protein of the glycoconjugate is the A chain ofCRM197 obtained via expression by genetically recombinant E. coli (see, e.g., CN103495161).In other embodiments, the carrier protein of the glycoconjugate is SCP (Streptococcal C5aPeptidase). Two important species of β-hemolytic streptococci, Streptococcus pyogenes (group AStreptococcus, GAS) and Streptococcus agalactiae (group B Streptococcus, GBS), which cause a variety ofserious human infections that range from mild cases of pharyngitis and impetigo to serious invasive diseases such as necrotizing fasciitis (GAS) and neonatal sepsis (GBS) have developed a way to defeat this immune response. All human isolates of β-hemolytic streptococci, including GAS and GBS, produce a highly conserved cell-wall protein SCP (Streptococcal C5a Peptidase) that specifically inactivates C5a. Thescp genes from GAS and GBS encode a polypeptide containing between 1,134 and 1,181 amino acids(Brown et al., PNAS, 2005, vol.102, no.51 pages 18391–18396). The first 31 residues are the export signal pre-sequence and are removed upon passing through the cytoplasmic membrane. The next 68 residues serve as a pro-sequence and must be removed to produce active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting with Lys-1034, are four consecutive 17-residue motifs followed by a cell sorting and cell-wall attachment signal. This combined signal is composed of a 20-residue hydrophilic sequence containing an LPTTND sequence, a 17-residue hydrophobic sequence, and a short basic carboxyl terminus. SCP can be divided into domains (see, e.g., figure 1B of Brown et al., PNAS, 2005, vol.102, no.51 pages 18391–18396). These domains are the Pre / Pro domain (which comprises the export signal pre- sequence (commonly the first 31 residues) and the pro-sequence (commonly the next 68 residues)), the protease domain (which is splitted in two part (protease part 1 commonly residues 89–333 / 334 and protease domain part 2 and commonly residues 467 / 468–583 / 584), the protease-associated domain (PA domain) (commonly residues 333 / 334–467 / 468), 3 fibronectin type III (Fn) domains (Fn1, commonly residues 583 / 584–712 / 713; Fn2, commonly residues 712 / 713–928 / 929 / 930; commonly Fn3, residues 929 / 930-1029 / 1030 / 1031) and a cell wall anchor domain (commonly redisues 1029 / 1030 / 1031 to the C- terminus). In some embodiments, the carrier protein of the glycoconjugate is an SCP from GBS (SCPB). Examples of SCPB include SEQ ID NO:3 of WO 97 / 26008 and SEQ ID NO:3 of WO00 / 34487. In other embodiments, the carrier protein of the glycoconjugate is an SCP from GAS (SCPA). Examples of SCPA include SEQ ID NO:1 and SEQ ID NO:2 of WO97 / 26008, and SEQ ID NOs: 1, 2 and 23 of WO00 / 34487. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP. In other embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP from GBS (SCPB). In other embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP from GAS (SCPA). In some embodiments, the carrier protein of the glycoconjugate is a fragment of an SCP.In some embodiments, the carrier protein of the glycoconjugate is a fragment of an SCPA. In someembodiments, the carrier protein of the glycoconjugate is a fragment of an SCPB. In some embodiments, the carrier protein of the glycoconjugate is a fragment of an SCP which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCP. In some embodiments, the carrier protein is an enzymatically inactive fragment of an SCP which comprises the protease domain, the protease-associated domain (PA domain) and 2 of the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro- sequence, or the cell wall anchor domain. In some embodiments, the enzymatically inactive fragment of SCP comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCPA. In some embodiments, the enzymatically inactive fragment of an SCPA comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPB. In some embodiments, the enzymatically inactive fragment of SCPB comprises theprotease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains,but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain. In some embodiments, the enzymatic activity of SCP is inactivated by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. The numbers indicate the amino acid residue position in the peptidase according to the numbering of SEQ ID NO: 1 of WO00 / 34487. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPA where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPB where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement isH193A. In another embodiment, said replacement is N295A. In yet another embodiment, saidreplacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCP where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPA which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. In another embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPB which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least one amino acid of the wild type sequence. In some embodiments, said replacement of at least one amino acid is in the protease domain. In some embodiments, said replacement of at least one amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acid is in part 2 of the protease domain. In some embodiments, said replacement is selected from the groupconsisting of D130A, H193A, N295A and S512A. In some embodiments, said replacement is D130A. Inanother embodiment, said replacement is H193A. In another embodiment, said replacement is N295A. In yet another embodiment, said replacement is S512A. In some embodiments, the enzymatic activity of SCP is inactivated by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said at least 2 amino acids replacements areselected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said atleast 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In someembodiments, said replacement of at least 2 amino acids is in the protease domain. In someembodiments, said replacement of at least 2 amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acid is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPA where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acid is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPB where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acid is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCP where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acid is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acid is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPA which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acids is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPB which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 2 amino acids of the wild type sequence. In some embodiments, said replacement of at least 2 amino acids is in the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 2 amino acids is in part 2 of the protease domain. In some embodiments, said at least 2 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 2 amino acids replacements are D130A and H193A. In some embodiments, said at least 2 amino acids replacements are D130A and N295A. In some embodiments, said at least 2 amino acids replacements are D130A and S512A. In some embodiments, said at least 2 amino acids replacements are H193A and N295A. In some embodiments, said at least 2 amino acids replacements are H193A and S512A. In some embodiments, said at least 2 amino acids replacements are N295A and S512A. In some embodiments, the enzymatic activity of SCP is inactivated by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 aminoacids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. Therefore, in some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPA where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPB where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCP where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acid is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPA which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPB which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 3 amino acids of the wild type sequence. In some embodiments, said replacement of at least 3 amino acids is in the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 3 amino acids is in part 2 of the protease domain. In some embodiments, said at least 3 amino acids replacements are selected from the group consisting of D130A, H193A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and N295A. In some embodiments, said at least 3 amino acids replacements are D130A, H193A and S512A. In some embodiments, said at least 3 amino acids replacements are D130A, N295A and S512A. In some embodiments, said at least 3 amino acids replacements are H193A, N295A and S512A. In some embodiments, the enzymatic activity of SCP is inactivated by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. Therefore, in some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPA where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCPB where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of an SCP where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is inpart 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acid is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPA which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least one amino acids is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCPB which comprises the protease domain, the protease-associated domain (PA domain) and the 3 fibronectin type III (Fn) domains, but does not comprise any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain, where said inactivation is accomplished by replacing at least 4 amino acids of the wild type sequence. In some embodiments, said replacement of at least 4 amino acids is in the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 1 of the protease domain. In some embodiments, said replacement of at least 4 amino acids is in part 2 of the protease domain. In some embodiments, said at least 4 amino acids replacements are D130A, H193A, N295A and S512A. In a particular non-limiting embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 41: MAKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNH EAWRLTDKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKT AVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLAD YARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGV SIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQL TETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGK IALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAF ISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKP DIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMT PSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYV TDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKH FALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLE GFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEAN SDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESS EITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGT FLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTR WDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATS ATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTF TLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity with SEQ ID NO: 41. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity with SEQ ID NO: 41. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99% identity with SEQ ID NO: 41. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity with SEQ ID NO: 41. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity with SEQ ID NO: 41. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity with SEQ ID NO: 41. In a particular non-limiting embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP which comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 42: AKTADTPATSKATIRDLNDPSQVKTLQEKAGKGAGTVVAVIAAGFDKNH EAWRLTDKAKARYQSKEDLEKAKKEHGITYGEWVNDKVAYYHDYSKDGKT AVDQEHGTHVSGILSGNAPSETKEPYRLEGAMPEAQLLLMRVEIVNGLAD YARNYAQAIRDAINLGAKVINMSFGNAALAYANLPDETKKAFDYAKSKGV SIVTSAGNDSSFGGKTRLPLADHPDYGVVGTPAAADSTLTVASYSPDKQL TETVTVKTADQQDKEMPVLSTNRFEPNKAYDYAYANRGTKEDDFKDVKGK IALIERGDIDFKDKIAKAKKAGAVGVLIYDNQDKGFPIELPNVDQMPAAF ISRKDGLLLKDNPQKTITFNATPKVLPTASGTKLSRFSSWGLTADGNIKP DIAAPGQDILSSVANNKYAKLSGTAMSAPLVAGIMGLLQEQYETQYPDMT PSERLDLAKKVLMSSATALYDEDEKAYFSPRQQGAGAVDAKKASAATMYV TDKDNTSSKVHLNNVSDKFEVTVTVHNKSDKPQELYYQATVQTDKVDGKH FALAPKALYETSWQKITIPANSSKQVTVPIDASRFSKDLLAQMKNGYFLE GFVRFKQDPKKEELMSIPYIGFRGDFGNLSALEKPIYDSKDGSSYYHEAN SDAKDQLDGDGLQFYALKNNFTALTTESNPWTIIKAVKEGVENIEDIESS EITETIFAGTFAKQDDDSHYYIHRHANGKPYAAISPNGDGNRDYVQFQGT FLRNAKNLVAEVLDKEGNVVWTSEVTEQVVKNYNNDLASTLGSTRFEKTR WDGKDKDGKVVANGTYTYRVRYTPISSGAKEQHTDFDVIVDNTTPEVATS ATFSTEDRRLTLASKPKTSQPVYRERIAYTYMDEDLPTTEYISPNEDGTF TLPEEAETMEGATVPLKMSDFTYVVEDMAGNITYTPVTKLLEGHSNKPEQ In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 90% identity with SEQ ID NO: 42. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 95% identity with SEQ ID NO: 42. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99% identity with SEQ ID NO: 42. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.5% identity with SEQ ID NO: 42. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.8% identity with SEQ ID NO: 42. In a particular embodiment, the carrier protein of the glycoconjugate is an enzymatically inactive fragment of SCP consisting of a polypeptide having at least 99.85% identity with SEQ ID NO: 42.Methods of preparing bacterial capsular saccharide carrier protein glycoconjugatesIn some embodiments, glycoconjugates of the disclosure are prepared using a so-called “click”chemical reaction. Also disclosed are methods of making a glycoconjugate using such click chemicalreactions. In some embodiments, the click chemical reaction is an azide-alkyne cycloaddition reaction catalyzed by Cu+1ions in which an azido activated saccharide is reacted with an alkyne activated carrier protein, forming a glycoconjugate. In some embodiments, methods of the disclosure further comprise,prior to performing an azide-alkyne cycloaddition reaction, preparing azido activated saccharide and / oralkyne activated carrier protein to be used as reactants in such reactions. Sizing capsular saccharides before conjugation In some embodiments, to generate glycoconjugates with advantageous filterability characteristics, immunogenicity and / or yields, sizing of the saccharide to a target molecular weight rangeis performed prior to the conjugation to a carrier protein. Advantageously, the size of the purified capsularsaccharide is reduced while preserving critical features of the structure of the polysaccharide. Mechanical or chemical sizing may be employed. In some embodiments, the size of the purified capsular saccharide is reduced by chemical hydrolysis. Chemical hydrolysis maybe conducted using a mild acid (e.g., acetic acid, formic acid, propanoic acid). In some embodiments, chemical hydrolysis is conducted using formic acid. In an embodiement, chemical hydrolysis is conducted using propanoic acid. In some embodiments, chemical hydrolysis is conducted using acetic acid. Chemical hydrolysis may also be conducted using a diluted strong acid (such as diluted hydrochloric acid, diluted sulfuric acid, diluted phosphoric acid, diluted nitric acid, or diluted perchloric acid). In some embodiments, chemical hydrolysis is conducted using diluted hydrochloric acid. In some embodiments, chemical hydrolysis is conducted using diluted sulfuric acid. In some embodiments, chemical hydrolysis is conducted using diluted phosphoric acid. In some embodiments, chemical hydrolysis is conducted using diluted nitric acid. In some embodiments, chemical hydrolysis is conducted using diluted perchloric acid. The size of the purified capsular saccharide can also be reduced by mechanical homogenization. In some embodiments, the size of the purified capsular saccharide is reduced by high pressure homogenization. High pressure homogenization achieves high shear rates by pumping the process stream through a flow path with sufficiently small dimensions. The shear rate is increased by using a larger applied homogenization pressure, and exposure time can be increased by recirculating the feed stream through the homogenizer. The high-pressure homogenization process can be appropriate for reducing the size of the purified capsular saccharide while preserving the structural features of the saccharide. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 10 kDa and 1000 kDa. In some embodiments, the isolated capsular saccharide is sized toa weight average molecular weight between 50 kDa and 500 kDa. In some embodiments, the isolatedcapsular saccharide is sized to a weight average molecular weight between 50 kDa and 400 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 50 kDa and 250 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 250 kDa and 1000 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 250 kDa and 500 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 250 kDa and 400 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight between 200 kDa and 800 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 250 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 300 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 350 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 400 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 450 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 500 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 550 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 600 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 700 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 800 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 900 kDa. In some embodiments, the isolated capsular saccharide is sized to a weight average molecular weight of about 1000 kDa. In some embodiments, the isolated capsular saccharide is not sized before its use in preparing a glycoconjugate as described herein. Preparing azido activated saccharide reactantIn some embodiments, an azido activated saccharide is prepared by reacting an isolated capsularsaccharide with a carbonic acid derivative and an azido linker in an aprotic solvent. After reacting thesaccharide in this manner, it can be said to be “activated” and can be referred to as an “activatedsaccharide” or “azido activated saccharide”. Before preparing activated saccharide, the saccharide to beactivated can be sized to a target molecular weight (Mw) range as described elsewhere herein. Azido linker In some embodiments, an azido linker for use in preparing an activated saccharide of thedisclosure is a compound of formula (I): (I),wherein X is selected from the group consisting of CH2(CH2)n, (CH2CH2O)mCH2CH2, NHCO(CH2)n,NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n and O(CH2CH2O)mCH2CH2; where n is selected from 1 to 10 and m isselected from 1 to 4. In some embodiments, an azido linker is a compound of formula (I), wherein X is CH2(CH2)n, and nis selected from 1 to 10. In some embodiments, n is selected from 1 to 5. In some embodiments, n isselected from 1 to 4. In some embodiments, n is selected from 1 to 3. In some embodiments, n is selectedfrom 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment,n is 3. In yet a further embodiment, n is 4. In yet a further embodiment, n is 5. In yet a further embodiment,n is 6. In yet a further embodiment, n is 7. In yet a further embodiment, n is 8. In yet a further embodiment, n is 9. In yet a further embodiment, n is 10. In some embodiments, an azido linker is a compound of formula (I), wherein X is(CH2CH2O)mCH2CH2, wherein m is selected from 1 to 4. In some embodiments, m is selected from 1 to 3.In some embodiments, m is selected from 1 to 2. In a particular embodiment, m is 1. In anotherembodiment, m is 2. In yet another embodiment, m is 3. In yet a further embodiment, m is 4.In some embodiments, an azido linker is a compound of formula (I), whereinin X is NHCO(CH2)n,and n is selected from 1 to 10. In some embodiments, n is selected from 1 to 5. In some embodiments, n is selected from 1 to 4. In some embodiments, n is selected from 1 to 3. In some embodiments, n is selected from 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet a further embodiment, n is 4. In yet a further embodiment, n is 5. In yet a further embodiment, n is 6. In yet a further embodiment, n is 7. In yet a further embodiment, n is 8. In yet a further embodiment, n is 9. In yet a further embodiment, n is 10. In some embodiments, an azido linker is a compound of formula (I), wherein X isNHCO(CH2CH2O)mCH2CH2, where m is selected from 1 to 4. In some embodiments, m is selected from 1 to3. In some embodiments, m is selected from 1 to 2. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In yet a further embodiment, m is 4. In some embodiments, an azido linker is a compound of formula (I), wherein X is OCH2(CH2)n, andn is selected from 1 to 10. In some embodiments, n is selected from 1 to 5. In some embodiments, n is selected from 1 to 4. In some embodiments, n is selected from 1 to 3. In some embodiments, n is selected from 1 to 2. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet a further embodiment, n is 4. In yet a further embodiment, n is 5. In yet a further embodiment, n is 6. In yet a further embodiment, n is 7. In yet a further embodiment, n is 8. In yet a further embodiment, n is 9. In yet a further embodiment, n is 10. In some embodiments, an azido linker is a compound of formula (I), wherein X isO(CH2CH2O)mCH2CH2, where m is selected from 1 to 4. In some embodiments, m is selected from 1 to 3. In some embodiments, m is selected from 1 to 2. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In yet a further embodiment, m is 4. In some embodiments, an azido linker for use in preparing an activated saccharide of thedisclosure is a compound of formula (II): (II). In some embodiments, an azido linker is 3-azido-propylamine.Carbonic acid derivative In some embodiments, a carbonic acid derivative for use in preparing an activated saccharide of the disclosure is selected from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di- (1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate, others being possible. In some embodiments, a carbonic acid derivative is 1,1'-carbonyldiimidazole (CDI). In another embodiment, a carbonic acid derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT). In another embodiment, said carbonic a derivative is disuccinimidyl carbonate (DSC). In yet a further embodiment, a carbonic acid derivative is N-hydroxysuccinimidyl chloroformate. In some embodiments, the step of preparing an activated saccharide comprises reacting thesaccharide with a carbonic acid derivative followed by reacting the carbonic acid derivative-activatedsaccharide with an azido linker in an aprotic solvent to produce an azido activated saccharide. In someembodiments such step comprises reacting the saccharide with an amount of carbonic acid derivativethat is between 0.01-10 molar equivalent to the amount of saccharide present in the reaction mixture. Insome embodiments such step comprises reacting the saccharide with an amount of carbonic acidderivative that is between 0.05-10 molar equivalent to the amount of saccharide present in the reactionmixture. In some embodiments such step comprises reacting the saccharide with an amount of carbonicacid derivative that is between 0.1-10 molar equivalent to the amount of saccharide present in thereaction mixture. In some embodiments such step comprises reacting the saccharide with an amount ofcarbonic acid derivative that is between 0.2-10 molar equivalent to the amount of saccharide present inthe reaction mixture. In some embodiments such step comprises reacting the saccharide with an amountof carbonic acid derivative that is between 0.3-10 molar equivalent to the amount of saccharide presentin the reaction mixture. In some embodiments such step comprises reacting the saccharide with anamount of carbonic acid derivative that is between 0.4-10 molar equivalent to the amount of saccharidepresent in the reaction mixture. In some embodiments such step comprises reacting the saccharide withan amount of carbonic acid derivative that is between 0.5-10 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative that is between 0.8-10 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 1-10 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 2-10 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 3-10 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 5-10 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.01-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.05-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.1-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.2-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.3-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.4-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.5-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.8-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 1-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 2-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 3-5 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.01-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.05-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.1-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.2-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.3-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.4-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.5-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.8-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 1-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 2-3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.01-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.05-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.1-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.2-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.3-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.4-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.5-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.8-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 1-2 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.01-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.05-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.1-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.2-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.3-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.4-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.5-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.8-1 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.01-0.5 molar equivalent tothe amount of saccharide present in the reaction mixture. In some embodiments such step comprisesreacting the saccharide with an amount of carbonic acid derivative that is between 0.05-0.5 molarequivalent to the amount of saccharide present in the reaction mixture. In some embodiments such stepcomprises reacting the saccharide with an amount of carbonic acid derivative that is between 0.1-0.5molar equivalent to the amount of saccharide present in the reaction mixture. In some embodiments suchstep comprises reacting the saccharide with an amount of carbonic acid derivative that is between 0.2-0.5 molar equivalent to the amount of saccharide present in the reaction mixture. In some embodimentssuch step comprises reacting the saccharide with an amount of carbonic acid derivative that is between0.3-0.5 molar equivalent to the amount of saccharide present in the reaction mixture. In someembodiments such step comprises reacting the saccharide with an amount of carbonic acid derivativethat is between 0.4-0.5 molar equivalent to the amount of saccharide present in the reaction mixture. Insome embodiments such step comprises reacting the saccharide with an amount of carbonic acidderivative that is between 0.01-0.4 molar equivalent to the amount of saccharide present in the reactionmixture. In some embodiments such step comprises reacting the saccharide with an amount of carbonicacid derivative that is between 0.05-0.4 molar equivalent to the amount of saccharide present in thereaction mixture. In some embodiments such step comprises reacting the saccharide with an amount ofcarbonic acid derivative that is between 0.1-0.4 molar equivalent to the amount of saccharide present inthe reaction mixture. In some embodiments such step comprises reacting the saccharide with an amountof carbonic acid derivative that is between 0.2-0.4 molar equivalent to the amount of saccharide presentin the reaction mixture. In some embodiments such step comprises reacting the saccharide with anamount of carbonic acid derivative that is between 0.3-0.4 molar equivalent to the amount of saccharidepresent in the reaction mixture. In some embodiments such step comprises reacting the saccharide withan amount of carbonic acid derivative that is between 0.01-0.3 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative that is between 0.05-0.3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.1-0.3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative that is between 0.2-0.3 molar equivalent to theamount of saccharide present in the reaction mixture. In some embodiments such step comprises reactingthe saccharide with an amount of carbonic acid derivative of about 0.01 molar equivalent to the amountof saccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.05 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.08 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.1 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.2 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.3 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.4 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 0.5 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 1 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 2 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 3 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 4 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 5 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 8 molar equivalent to the amount ofsaccharide present in the reaction mixture. In some embodiments such step comprises reacting thesaccharide with an amount of carbonic acid derivative of about 10 molar equivalent to the amount ofsaccharide present in the reaction mixture. Aprotic solvent In some embodiments, the step of reacting the isolated saccharide with a carbonic acid derivativeis carried out in an aprotic solvent. In some embodiments the isolated saccharide is reacted with acarbonic acid derivative in a solution comprising, consisting essentially of, or consisting ofdimethylsulphoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, orhexamethylphosphoramide (HMPA). In some embodiments the isolated saccharide is reacted with acarbonic acid derivative in a solution consisting essentially of dimethylformamide (DMF). In someembodiments the isolated saccharide is reacted with a carbonic acid derivative in a solution consistingessentially of dimethylsulphoxide (DMSO). In some embodiments, the carbonic acid derivative is CDI and the aprotic solvent is dimethylsulphoxide (DMSO). In some embodiments, the carbonic acid derivative is CDI and the aprotic solvent is anhydrous DMSO. It has been surprisingly found that reacting the isolated saccharide with CDI in solution with amoisture level of about 0.1% to 1% volume / volume (v / v) reduces the frequency of unwanted sidereactions. Therefore, in some embodiments the isolated saccharide is reacted with CDI in an aproticsolvent comprising 0.1% to 1% (v / v) water. In some embodiments the isolated saccharide is reacted withCDI in an aprotic solvent comprising 0.1% to 0.8% (v / v) water. In some embodiments the isolatedsaccharide is reacted with CDI in an aprotic solvent comprising 0.1% to 0.5% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in an aprotic solvent comprising 0.1% to 0.4%(v / v) water. In some embodiments the isolated saccharide is reacted with CDI in an aprotic solventcomprising 0.1% to 0.3% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI inan aprotic solvent comprising 0.1% to 0.2% (v / v) water. In some embodiments the isolated saccharide isreacted with CDI in an aprotic solvent comprising 0.2% to 1% (v / v) water. In some embodiments theisolated saccharide is reacted with CDI in an aprotic solvent comprising 0.2% to 0.8% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in an aprotic solvent comprising 0.2% to 0.5%(v / v) water. In some embodiments the isolated saccharide is reacted with CDI in an aprotic solventcomprising 0.2% to 0.4% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI inan aprotic solvent comprising 0.2% to 0.3% (v / v) water. In some embodiments the isolated saccharide isreacted with CDI in an aprotic solvent comprising 0.3% to 0.8% (v / v) water. In some embodiments theisolated saccharide is reacted with CDI in an aprotic solvent comprising 0.3% to 0.5% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in an aprotic solvent comprising 0.3% to 0.4%(v / v) water. In some embodiments the isolated saccharide is reacted with CDI in an aprotic solventcomprising about 0.1% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI inan aprotic solvent comprising about 0.2% (v / v) water. In some embodiments the isolated saccharide isreacted with CDI in an aprotic solvent comprising about 0.3% (v / v) water. In some embodiments theisolated saccharide is reacted with CDI in an aprotic solvent comprising about 0.4% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in an aprotic solvent comprising about 0.5% (v / v)water. In some embodiments the isolated saccharide is reacted with CDI in an aprotic solvent comprisingabout 0.6% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI in an aproticsolvent comprising about 0.7% (v / v) water. In some embodiments the isolated saccharide is reacted withCDI in an aprotic solvent comprising about 0.8% (v / v) water. In some embodiments the isolated saccharideis reacted with CDI in an aprotic solvent comprising about 0.9% (v / v) water.In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising 0.1% to 1%(v / v) water. In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising 0.1%to 0.8% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising0.1% to 0.5% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI in DMSOcomprising 0.1% to 0.4% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI inDMSO comprising 0.1% to 0.3% (v / v) water. In some embodiments the isolated saccharide is reacted withCDI in DMSO comprising 0.1% to 0.2% (v / v) water. In some embodiments the isolated saccharide isreacted with CDI in DMSO comprising 0.2% to 1% (v / v) water. In some embodiments the isolatedsaccharide is reacted with CDI in DMSO comprising 0.2% to 0.8% (v / v) water. In some embodiments theisolated saccharide is reacted with CDI in DMSO comprising 0.2% to 0.5% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in DMSO comprising 0.2% to 0.4% (v / v) water.In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising 0.2% to 0.3% (v / v)water. In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising 0.3% to0.8% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI in DMSO comprising0.3% to 0.5% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI in DMSOcomprising 0.3% to 0.4% (v / v) water. In some embodiments the isolated saccharide is reacted with CDI inDMSO comprising about 0.1% (v / v) water. In some embodiments the isolated saccharide is reacted withCDI in DMSO comprising about 0.2% (v / v) water. In some embodiments the isolated saccharide is reactedwith CDI in DMSO comprising about 0.3% (v / v) water. In some embodiments the isolated saccharide isreacted with CDI in DMSO comprising about 0.4% (v / v) water. In some embodiments the isolatedsaccharide is reacted with CDI in DMSO comprising about 0.5% (v / v) water. In some embodiments theisolated saccharide is reacted with CDI in DMSO comprising about 0.6% (v / v) water. In some embodimentsthe isolated saccharide is reacted with CDI in DMSO comprising about 0.7% (v / v) water. In someembodiments the isolated saccharide is reacted with CDI in DMSO comprising about 0.8% (v / v) water. Insome embodiments the isolated saccharide is reacted with CDI in DMSO comprising about 0.9% (v / v)water. Quenching unreacted carbonic acid derivative In some embodiments, after reacting the saccharide with the carbonic acid derivative (such asCDI), unreacted free carbonic acid derivative in the reaction mixture can be quenched by the addition ofwater before the addition of the azido linker. Accordingly, in some embodiments, saccharide activationwith carbonic acid derivative is followed by the addition of water. In some embodiments, water is addedto bring the total water content in the reaction mixture to between about 1% to about 10% (v / v). In some embodiments, water is added to bring the total water content in the mixture to between about 1.2% to about 8% (v / v). In some embodiments, water is added to bring the total water content in the mixture to between about 1.5% to about 5% (v / v). In some embodiments, water is added to bring the total water content in the mixture to between about 1.5% to about 3% (v / v). In some embodiments, water is added to bring the total water content in the mixture to between about 1.5% to about 2.5% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 1 % (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 1.2% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 1.4% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 1.5% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 2% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 2.5% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 3% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 5% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 7% (v / v). In some embodiments, water is added to bring the total water content in the mixture to about 10% (v / v). Reacting carbonic acid derivative-activated saccharide with azido linkerIn some embodiments, after unreacted free carbonic acid derivative in the reaction mixture is quenched by the addition of water, carbonic acid derivative-activated saccharide is reacted with an azidolinker. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with an amountof azido linker that is between 0.01-10 molar equivalents to the number of polysaccharide repeat units ofthe activated saccharide (molar equivalent of RU). In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.01-8 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.01-5 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.01-4 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.01-3 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.01-2 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.01-1 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.01-0.5 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.01-0.1 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.05-10 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.05-8 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.05-5 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.05-4 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.05-3 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.05-2 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.05-1 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.05-0.5 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.05-0.1 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.1-10 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.1-8 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.1-5 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.1-4 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.1-3 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.1-2 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.1-1 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.1-0.5 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.5-10 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.5-8 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.5-5 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 0.5-4 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 0.5-3 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 0.5-2 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 0.5-1 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 1-10 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 1-8 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 1-5 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 1-4 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 1-3 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 1-2 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 2-10 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 2-8 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 2-5 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 2-4 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 2-3 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 3-10 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 3-8 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 3-5 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 3-4 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 4-10 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 4-8 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is between 4-5 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is between 5-10 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is between 5-8 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is between 8-10 molar equivalents to the number of polysaccharide repeatunits of the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharideis reacted with an amount of azido linker that is about 0.01 molar equivalents to the number ofpolysaccharide repeat units of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is about 0.05 molarequivalents to the number of polysaccharide repeat units of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is about 0.1 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is about 0.5 molar equivalents to the number of polysaccharide repeat unitsof the activated saccharide. In some embodiments, the carbonic acid derivative-activated saccharide isreacted with an amount of azido linker that is about 1 molar equivalents to the number of polysacchariderepeat units of the activated saccharide. In some embodiments, the carbonic acid derivative-activatedsaccharide is reacted with an amount of azido linker that is about 2 molar equivalents to the amount ofpolysaccharide repeat unit of the activated saccharide. In some embodiments, the carbonic acidderivative-activated saccharide is reacted with an amount of azido linker that is about 3 molar equivalentsto the amount of polysaccharide repeat unit of the activated saccharide. In some embodiments, thecarbonic acid derivative-activated saccharide is reacted with an amount of azido linker that is about 4molar equivalents to the amount of polysaccharide repeat unit of the activated saccharide. In someembodiments, the carbonic acid derivative-activated saccharide is reacted with an amount of azido linkerthat is about 5 molar equivalents to the amount of polysaccharide repeat unit of the activated saccharide.In some embodiments, the carbonic acid derivative-activated saccharide is reacted with an amount ofazido linker that is about 8 molar equivalents to the amount of polysaccharide repeat unit of the activatedsaccharide. In some embodiments, the carbonic acid derivative-activated saccharide is reacted with anamount of azido linker that is about 10 molar equivalents to the amount of polysaccharide repeat unit ofthe activated saccharide. In any of the foregoing embodiments, the saccharide is activated by reacting the saccharide with the carbonic acid derivative CDI or the carbonic acid derivative CDT. Degree of activation of azido activated saccharide Once prepared, azido activated saccharides formed by reacting a capsular saccharide with a carbonic acid derivative and an azido linker can be characterized by their degree of activation, which is defined as the proportion of polysaccharide repeat units in the saccharide that are linked to an azido linker. Degree of activation of azido activated saccharide can be expressed in a variety of ways. Forexample, in some embodiments, degree of activation can be expressed as a single number which standsfor the denominator of the ratio of the number of azido linkers per number of repeat units of an azidoactivated saccharide, where the ratio is simplified so that the numerator is 1. Alternatively, in other embodiments, degree of activation can be expressed as the percentage of repeat units in the azido activated saccharide that are linked to an azido linker. For example, if an azido activated saccharide on average has 1 azido linker for every 5 repeat units, the degree of activation of said azido activated saccharide can either be expressed as 5 or as 20%.In some embodiments, after reacting the carbonic acid derivative-activated saccharide with theazido linker, the degree of activation of the azido activated saccharide is between 0.5 to 50%. In someembodiments the degree of activation of the activated saccharide is between 1 to 30%. In anotherembodiment the degree of activation of the activated saccharide is between 2 to 25%. In anotherembodiment the degree of activation of the activated saccharide is between 3 to 20%. In anotherembodiment the degree of activation of the activated saccharide is between 3 to 15%. In anotherembodiment the degree of activation of the activated saccharide is between 4 to 15%. In someembodiments the degree of activation of the activated saccharide is between 1 to 6%. In someembodiments the degree of activation of the activated saccharide is between 3 to 6%. In someembodiments the degree of activation of the activated saccharide is between 10 to 15%. In someembodiments the degree of activation of the activated saccharide is about 1%. In some embodiments thedegree of activation of the activated saccharide is about 2%. In some embodiments the degree ofactivation of the activated saccharide is about 3%. In some embodiments the degree of activation of theactivated saccharide is about 4%. In some embodiments the degree of activation of the activatedsaccharide is about 5%. In some embodiments the degree of activation of the activated saccharide is about6%. In some embodiments the degree of activation of the activated saccharide is about 7%. In someembodiments the degree of activation of the activated saccharide is about 8%. In some embodiments thedegree of activation of the activated saccharide is about 9%. In some embodiments the degree ofactivation of the activated saccharide is about 10%. In some embodiments the degree of activation of theactivated saccharide is about 11%. In some embodiments the degree of activation of the activatedsaccharide is about 12%. In some embodiments the degree of activation of the activated saccharide isabout 13%. In some embodiments the degree of activation of the activated saccharide is about 14%. Insome embodiments the degree of activation of the activated saccharide is about 15%. In someembodiments the degree of activation of the activated saccharide is about 16%. In some embodimentsthe degree of activation of the activated saccharide is about 17%. In some embodiments the degree ofactivation of the activated saccharide is about 18%. In some embodiments the degree of activation of theactivated saccharide is about 19%. In some embodiments the degree of activation of the activatedsaccharide is about 20%. Preparing alkyne activated carrier protein reactantIn some embodiments, an alkyne functionalized carrier protein is prepared by reacting a carrierprotein with an agent (reactant) bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group,where the NHS moiety reacts with amino groups in the carrier protein to form an amide linkage. Afterreacting the carrier protein in this manner, it can be said to be “activated” and can be referred to as an“activated carrier” or an “alkyne functionalized carrier protein”.Agents having NHS moiety and alkyne groupIn some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkynegroup for use in preparing an alkyne functionalized carrier protein of the disclosure is an agent bearing anN-hydroxysuccinimide (NHS) moiety and a terminal alkyne. In some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group is an agent bearing an N-hydroxysuccinimide (NHS) moiety and a cycloalkyne. In some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a compound of formula (III): where X is selected from the group consisting of CH2O(CH2)nCH2C=O and CH2O(CH2CH2O)m(CH2)nCH2C=O, where n is selected from 0 to 10 and m is selected from 0 to 4. In some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkynegroup is a compound of formula (III), wherein X is CH2O(CH2)nCH2C=O, where n is selected from 0 to 10.In some embodiments, n is selected from 0 to 5. In some embodiments, n is selected from 0 to 4. In some embodiments, n is selected from 0 to 3. In some embodiments, n is selected from 0 to 2. In aparticular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yetanother embodiment, n is 3. In yet a further embodiment, n is 4. In yet a further embodiment, n is 5. In yet a further embodiment, n is 6. In yet a further embodiment, n is 7. In yet a further embodiment, n is 8. In yet a further embodiment, n is 9. In yet a further embodiment, n is 10. In some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkynegroup is a compound of formula (III), wherein X is CH2O(CH2CH2O)m(CH2)nCH2C=O, where n is selected from0 to 10 and m is selected from 0 to 4. In some embodiments, n is selected from 0 to 5. In some embodiments, n is selected from 0 to 4. In some embodiments, n is selected from 0 to 3. In some embodiments, n is selected from 0 to 2. In a particular embodiment, n is 0. In a particular embodiment, n is 1. In another embodiment, n is 2. In yet another embodiment, n is 3. In yet a further embodiment, n is 4. In yet a further embodiment, n is 5. In yet a further embodiment, n is 6. In yet a further embodiment, n is 7. In yet a further embodiment, n is 8. In yet a further embodiment, n is 9. In yet a further embodiment, n is 10. In some embodiments, m is selected from 0 to 3. In some embodiments, m is selected from 0 to 2. In a particular embodiment, m is 1. In a particular embodiment, m is 1. In another embodiment, m is 2. In yet another embodiment, m is 3. In yet a further embodiment, m is 4. In some embodiments, n is selected from 0 to 5 and m is selected from 0 to 3. In some embodiments, n is selected from 0 to 5 and m is selected from 0 to 2. In some embodiments, n is selected from 0 to 4 and m is selected from 0 to 3. In some embodiments, n is selected from 0 to 4 and m is selected from 0 to 2. In some embodiments, n is selected from 0 to 3 and m is selected from 0 to 3. In some embodiments, n is selected from 0 to 3 and m is selected from 0 to 2. In some embodiments, n is selected from 0 to 2 and m is selected from 0 to 3. In some embodiments, n is selected from 0 to 2 and m is selected from 0 to 2. In some embodiments, n is selected from 0 to 1 and m is selected from 0 to 3. In some embodiments, n is selected from 0 to 1 and m is selected from 0 to 2. In some embodiments, n is 0 and m is 0. In some embodiments, n is 1 and m is 0. In some embodiments, n is 2 and m is 0. In some embodiments, n is 3 and m is 0. In some embodiments, n is 4 and m is 0. In some embodiments, n is 5 and m is 0. In some embodiments, n is 6 and m is 0. In some embodiments, n is 7 and m is 0. In some embodiments, n is 8 and m is 0. In some embodiments, n is 9 and m is 0. In some embodiments, n is 10 and m is 0. In some embodiments, n is 0 and m is 1. In some embodiments, n is 1 and m is 1. In some embodiments, n is 2 and m is 1. In some embodiments, n is 3 and m is 1. In some embodiments, n is 4 and m is 1. In some embodiments, n is 5 and m is 1. In some embodiments, n is 6 and m is 1. In some embodiments, n is 7 and m is 1. In some embodiments, n is 8 and m is 1. In some embodiments, n is 9 and m is 1. In some embodiments, n is 10 and m is 1. In some embodiments, n is 0 and m is 2. In some embodiments, n is 1 and m is 2. In some embodiments, n is 2 and m is 2. In some embodiments, n is 3 and m is 2. In some embodiments, n is 4 and m is 2. In some embodiments, n is 5 and m is 2. In some embodiments, n is 6 and m is 2. In some embodiments, n is 7 and m is 2. In some embodiments, n is 8 and m is 2. In some embodiments, n is 9 and m is 2. In some embodiments, n is 10 and m is 2. In some embodiments, n is 0 and m is 3. In some embodiments, n is 1 and m is 3. In some embodiments, n is 2 and m is 3. In some embodiments, n is 3 and m is 3. In some embodiments, n is 4 and m is 3. In some embodiments, n is 5 and m is 3. In some embodiments, n is 6 and m is 3. In some embodiments, n is 7 and m is 3. In some embodiments, n is 8 and m is 3. In some embodiments, n is 9 and m is 3. In some embodiments, n is 10 and m is 3. In some embodiments, n is 0and m is 4. In some embodiments, n is 1 and m is 4. In some embodiments, n is 2 and m is 4. In someembodiments, n is 3 and m is 4. In some embodiments, n is 4 and m is 4. In some embodiments, n is 5 and m is 4. In some embodiments, n is 6 and m is 4. In some embodiments, n is 7 and m is 4. In some embodiments, n is 8 and m is 4. In some embodiments, n is 9 and m is 4. In some embodiments, n is 10 and m is 4. In some embodiments, an agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group is a compound of formula (IV): Reacting carrier protein with agent having NHS moiety and alkyne groupIn some embodiments, to prepare alkyne activated (or alkyne functionalized) carrier protein, acarrier protein is reacted with an amount of an agent bearing an N-hydroxysuccinimide (NHS) moiety andan alkyne group that is 0.1-10 molar equivalents to the lysines on the carrier. In some embodiments thecarrier protein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and analkyne group that is 0.5-10 molar equivalents to the lysines on the carrier. In some embodiments thecarrier protein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety andan alkyne group that is 1-10 molar equivalents to the lysines on the carrier. In some embodiments thecarrier protein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and analkyne group that is 1.5-10 molar equivalents to the lysines on the carrier. In some embodiments thecarrier protein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and analkyne group that is 2-10 molar equivalents to the lysines on the carrier. In some embodiments the carrierprotein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkynegroup that is 2.5-10 molar equivalents to the lysines on the carrier. In some embodiments the carrierprotein is reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkynegroup that is 3-10 molar equivalents to the lysines on the carrier. In some embodiments the carrier proteinis reacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne groupthat is 5-10 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein isreacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group thatis 7.5-10 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein isreacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group thatis 0.1-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein isreacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group thatis 0.5-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein isreacted with an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group thatis 1-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 5-7.5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reactedwith an amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-5 molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1-5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5-5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2-5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5-5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 3-5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2.5-3 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-2.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-2.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1-2.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5-2.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 2-2.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-2 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-2 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1-2 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1.5-2 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-1.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-1.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 1-1.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-1 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.5-1 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is 0.1-0.5 molarequivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted with anamount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 10molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 7.5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 3molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 2.5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 2molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 1.5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 1molar equivalent to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 0.5molar equivalents to the lysines on the carrier. In some embodiments the carrier protein is reacted withan amount of agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group that is about 0.1molar equivalents to the lysines on the carrier. Degree of activation of activated carrier protein Once prepared, alkyne functionalized carrier proteins formed by reacting carrier protein with the agent bearing an N-hydroxysuccinimide (NHS) moiety and an alkyne group can be characterized by theirdegree of activation, which is defined as the number of lysine residues in the carrier protein which are onaverage linked to the agent bearing the N-hydroxysuccinimide (NHS) moiety and alkyne group. In some embodiments, after preparing, the degree of activation of the activated carrier protein isbetween 1 and 50. In some embodiments, the carrier protein is CRM197, which contains a total of 39 lysineresidues. In said embodiments the degree of activation of the activated carrier may be between 1 to 30.In another embodiment the degree of activation of the activated carrier (CRM197) is between 5 to 20. Inanother embodiment the degree of activation of the activated carrier (CRM197) is between 9 to 18. Inanother embodiment the degree of activation of the activated carrier (CRM197) is between 8 to 11. Inanother embodiment the degree of activation of the activated carrier (CRM197) is between 15 to 20. Inanother embodiment the degree of activation of the activated carrier (CRM197) is about 5. In anotherembodiment the degree of activation of the activated carrier (CRM197) is about 6. In another embodiment the degree of activation of the activated carrier (CRM197) is about 7. In another embodiment the degree of activation of the activated carrier (CRM197) is about 8. In another embodiment the degree of activation of the activated carrier (CRM197) is about 9. In another embodiment the degree of activation of the activated carrier (CRM197) is about 10. In another embodiment the degree of activation of the activated carrier (CRM197) is about 11. In another embodiment the degree of activation of the activated carrier (CRM197) is about 12. In another embodiment the degree of activation of the activated carrier (CRM197) is about 13. In another embodiment the degree of activation of the activated carrier (CRM197) is about 14. In another embodiment the degree of activation of the activated carrier (CRM197) is about 15. In another embodiment the degree of activation of the activated carrier (CRM197) is about 16. In another embodiment the degree of activation of the activated carrier (CRM197) about 17. In another embodiment the degree of activation of the activated carrier (CRM197) is about 18. In another embodiment the degree of activation of the activated carrier (CRM197) about 19. In another embodiment the degree of activation of the activated carrier (CRM197) is about 20. In another embodiment the degree of activation of the activated carrier (CRM197) about 21. In another embodiment the degree of activation of the activated carrier (CRM197) is about 22. In another embodiment the degree of activation of the activated carrier (CRM197) about 23. In another embodiment the degree of activation of the activated carrier (CRM197) is about 24. In another embodiment the degree of activation of the activated carrier (CRM197) is about 25. In some embodiments, the carrier protein is SCP or a fragment thereof. In said embodiments thedegree of activation of the activated carrier may be between 1 to 50. In another embodiment the degreeof activation of the activated carrier (SCP) is between 5 to 50. In another embodiment the degree ofactivation of the activated carrier (SCP) is between 7 to 45. In another embodiment the degree of activation of the activated carrier (SCP) is between 5 to 15. In another embodiment the degree of activation of the activated carrier (SCP) is between 20 to 30. In another embodiment the degree of activation of the activated carrier (SCP) is between 30 to 50. In another embodiment the degree ofactivation of the activated carrier (SCP) is between 30 to 40. In another embodiment the degree ofactivation of the activated carrier (SCP) is between 10 to 40. In another embodiment the degree ofactivation of the activated carrier (SCP) is about 5. In another embodiment the degree of activation of the activated carrier (SCP) is about 7. In another embodiment the degree of activation of the activated carrier (SCP) is about 10. In another embodiment the degree of activation of the activated carrier (SCP) is about 13. In another embodiment the degree of activation of the activated carrier (SCP) is about 15. In another embodiment the degree of activation of the activated carrier (SCP) is about 20. In another embodiment the degree of activation of the activated carrier (SCP) is about 26. In another embodiment the degree ofactivation of the activated carrier (SCP) is about 30. In another embodiment the degree of activation ofthe activated carrier (SCP) is about 35. In another embodiment the degree of activation of the activated carrier (SCP) is about 37. In another embodiment the degree of activation of the activated carrier (SCP) is about 40. In another embodiment the degree of activation of the activated carrier (SCP) is about 45. In another embodiment the degree of activation of the activated carrier (SCP) is about 50. In some embodiments, the carrier protein is TT or a fragment thereof. In said embodiment thedegree of activation of the activated carrier may be between 1 to 30. In another embodiment the degreeof activation of the activated carrier (TT) is between 5 to 25. In another embodiment the degree of activation of the activated carrier (TT) is between 7 to 25. In another embodiment the degree of activationof the activated carrier (TT) is between 10 to 20. In another embodiment the degree of activation of theactivated carrier (TT) is about 5. In another embodiment the degree of activation of the activated carrier (TT) is about 7. In another embodiment the degree of activation of the activated carrier (TT) is about 10. In another embodiment the degree of activation of the activated carrier (TT) is about 12. In another embodiment the degree of activation of the activated carrier (TT) is about 15. In another embodiment the degree of activation of the activated carrier (TT) is about 20. In another embodiment the degree of activation of the activated carrier (TT) is about 25. In another embodiment the degree of activation of the activated carrier (TT) is about 30. Azide-alkyne cycloaddition reaction In some embodiments, the conjugation reaction (click chemical reaction) between the azidoactivated saccharide and the alkyne activated (functionalized) carrier protein forming the glycoconjugateis carried out in the presence of copper (I) as catalyst. Copper (I) catalyst can be provided in any wayknown in the art. In some embodiments, a Cu+2 inorganic or organic salt may be added directly to areaction mixture with the addition of another compound acting as a reducing agent. Non-limiting examples of Cu+2salts include CuSO4and Cu(OAc)2, whereas non-limiting examples of reducing agents include sodium ascorbate (NaAsc), hydrazine, and tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, a copper stabilizing agent, such as tris-(hydroxypropyltriazolylmethyl)amine (THPTA) or Tris(benzyltriazolylmethyl)amine (TBTA), may additionally be included in the reaction mixture to protectthe Cu(I) catalyst from oxidation once formed. In some embodiments, an agent may be included in thereaction mixture to prevent oxidation byproducts from reacting with and modifying carrier protein, an example being aminoguanidine. In some embodiments, azide-alkyne cycloaddition reactions which arecarried out using a Cu+2 salt (such as CuSO4) and a reducing agent (such as sodium ascorbate), optionallyfurther including a copper stabilizing agent (such as THPTA) and / or an agent to prevent undesired proteinmodification by reactive oxygen species (such as aminoguanidine), can be performed in water or anaqueous solvent or buffer.In other embodiments, a Cu(I) catalyst can be provided by adding a Cu+1inorganic or organic saltdirectly to the reaction mixture, optionally in the absence of a reducing agent. Non-limiting examples ofCu+1 salts include CuBr, CuI, CuOTf·C6H6 (OTf =trifluoromethanesulfonate), and [Cu(NCCH3)4][PF6]. In yetother embodiments, metallic copper can be provided along with addition of a oxidizing agent, such as an amine salt. Determining general conditions for azide-alkyne cycloaddition reactions useful herein, such asrelative amounts and concentrations of chemical agents used in the reaction, temperature, and time forthe reaction to proceed, as well as others, is within the knowledge of those of ordinarily skilled in the art. Additional information about click-chemical reactions useful in the methods described herein maybe found in, among other references, Agrahari, AK, et al., Cu(I)-Catalyzed Click Chemistry in Glycoscienceand Their Diverse Applications, Chemical Reviews 2021121 (13), 7638-7956, DOI:10.1021 / acs.chemrev.0c00920; Tiwari, VK, et al., Cu-Catalyzed Click Reaction in Carbohydrate ChemistryChemical Reviews 2016 116 (5), 3086-3240, DOI: 10.1021 / acs.chemrev.5b00408; Hein, CD, et al., ClickChemistry, A Powerful Tool for Pharmaceutical Sciences. Pharm Res 25, 2216–2230 (2008), https: / / doi.org / 10.1007 / s11095-008-9616-1. Cycloaddition reaction comprising plurality of capsular saccharide serotypes In some embodiments, the azide-alkyne cycloaddition reaction comprises azido activated capsularsaccharide of at least two structurally and / or antigenically distinct bacterial serotypes. In this way, glycoconjugates can be prepared comprising carrier protein to which two or more capsular saccharides of distinct serotype are simultaneously conjugated. In some embodiments, the azide-alkyne cycloaddition reaction comprises azido activated saccharide of at least two structurally and / or antigenically distinct serotypes of Streptococcus pneumoniae (S. pneumoniae) bacteria. Such azido activated saccharides can be added to the reaction mixture in anysuitable concentration or mass relative to each other, or relative to the carrier protein also added to thereaction mixture. In some embodiments, the azido activated saccharides of at least 2 distinct S.pneumoniae serotypes are added to the reaction mixture in approximately equal masses, such that theratio of the masses is approximately or about 1:1.In some embodiments, the reaction mixture comprises azido activated saccharides of 2 distinct S.pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1. In someembodiments, the reaction mixture comprises azido activated saccharides of 3 distinct S. pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1:1. In some embodiments,the reaction mixture comprises azido activated saccharides of 4 distinct S. pneumoniae serotypes, whichare added to the reaction mixture in a mass ratio of about 1:1:1:1. In some embodiments, the reactionmixture comprises azido activated saccharides of 5 distinct S. pneumoniae serotypes, which are added tothe reaction mixture in a mass ratio of about 1:1:1:1:1. In some embodiments, the reaction mixturecomprises azido activated saccharides of 6 distinct S. pneumoniae serotypes, which are added to thereaction mixture in a mass ratio of about 1:1:1:1:1:1. In some embodiments, azido activated saccharides of at least 2 distinct S. pneumoniae serotypesare added to the reaction mixture in a mass ratio which differs from 1:1. Thus, for example, in someembodiments, azido activated saccharides of at least 2 distinct S. pneumoniae serotypes can be added tothe reaction mixture in a mass ratio of x:y, where the antecedent value corresponds to the relative massof first serotype and the consequent value corresponds to the relative mass of the at least secondserotype, and where x=1 and y is any value greater than 1 (>1), for example at least or about 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated saccharides of 2 distinct S.pneumoniae serotypes which are added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values,such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated saccharides of 3 distinct S.pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1:n, where thefirst 2 values in the series represent the relative masses of 2 of the azido activated saccharide serotypesand the third value, n, represents the relative mass of the third which is added, where n is any value >1,for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumeratedvalues, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated saccharides of 4 distinct S.pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1:1:n, wherethe first 3 values in the series represent the relative masses of 3 of the azido activated saccharideserotypes and the fourth value, n, represents the relative mass of the fourth which is added, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specificallyenumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated saccharides of 5 distinct S.pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1:1:1:n, wherethe first 4 values in the series represent the relative masses of 4 of the azido activated saccharideserotypes and the fifth value, n, represents the relative mass of the fifth which is added, where n is anyvalue >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated saccharides of 6 distinct S.pneumoniae serotypes, which are added to the reaction mixture in a mass ratio of about 1:1:1:1:1:n,where the first 5 values in the series represent the relative masses of 5 of the azido activated saccharideserotypes and the sixth value, n, represents the relative mass of the sixth which is added, where n is anyvalue >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 1, and the atleast second serotype is selected from the group consisting of serotypes 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 1 and 2, 1 and 3, 1 and 4, 1 and 5, 1 and 6A, 1 and 6B, 1 and 6C, 1 and 7C, 1 and 7F, 1 and 8, 1 and 9V, 1 and 9N, 1 and 10A, 1 and 10B, 1 and 11A, 1 and 12F, 1 and 14, 1 and 15A, 1 and 15B, 1 and 15C, 1 and 16F, 1 and 17F, 1 and 18C, 1 and 19A, 1 and 19F, 1 and 20, 1 and 20B, 1 and 21, 1 and 22A, 1 and 22F, 1 and 23A, 1 and 23B, 1 and 23F, 1 and 24B, 1 and 24F, 1 and 27, 1 and 29, 1 and 31, 1 and 33B, 1 and 33F, 1 and 34, 1 and 35B, 1 and 35F, 1 and 38, 1 and 72, or 1 and 73. In some embodiments, the reaction mixture comprises azido activatedcapsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at leastthird, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and fromeach of said lower order numbered serotypes, and is selected from the group consisting of serotypes 2,3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of theforegoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotypecan be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequentvalues differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value>1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specificallyenumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 2, and the atleast second serotype is selected from the group consisting of serotypes 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 2 and 3, 2 and 4, 2 and 5, 2 and6A, 2 and 6B, 2 and 6C, 2 and 7C, 2 and 7F, 2 and 8, 2 and 9V, 2 and 9N, 2 and 10A, 2 and 10B, 2 and 11A,2 and 12F, 2 and 14, 2 and 15A, 2 and 15B, 2 and 15C, 2 and 16F, 2 and 17F, 2 and 18C, 2 and 19A, 2 and 19F, 2 and 20, 2 and 20B, 2 and 21, 2 and 22A, 2 and 22F, 2 and 23A, 2 and 23B, 2 and 23F, 2 and 24B, 2 and 24F, 2 and 27, 2 and 29, 2 and 31, 2 and 33B, 2 and 33F, 2 and 34, 2 and 35B, 2 and 35F, 2 and 38, 2 and 72, or 2 and 73. In some embodiments, the reaction mixture comprises azido activated capsularsaccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third,fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each ofsaid lower order numbered serotypes, and is selected from the group consisting of serotypes 1, 3, 4, 5,6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoingembodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be addedto the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less,or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for exampleat least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 3, and the atleast second serotype is selected from the group consisting of serotypes 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 3 and 4, 3 and 5, 3 and 6A, 3 and 6B, 3 and 6C, 3 and 7C, 3 and 7F, 3 and 8, 3 and 9V, 3 and 9N, 3 and 10A, 3 and 10B, 3 and 11A, 3 and 12F, 3 and 14, 3 and 15A, 3 and 15B, 3 and 15C, 3 and 16F, 3 and 17F, 3 and 18C, 3 and 19A, 3 and 19F, 3 and 20, 3 and 20B, 3 and 21, 3 and 22A, 3 and 22F, 3 and 23A, 3 and 23B, 3 and 23F, 3 and 24B, 3 and 24F, 3 and27, 3 and 29, 3 and 31, 3 and 33B, 3 and 33F, 3 and 34, 3 and 35B, 3 and 35F, 3 and 38, 3 and 72, or 3 and73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at leasta third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 4, 5, 6A, 6B, 6C, 7C, 7F,8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or anyvalue between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 4, and the atleast second serotype is selected from the group consisting of serotypes 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 4 and 5, 4 and 6A, 4 and 6B, 4 and 6C,4 and 7C, 4 and 7F, 4 and 8, 4 and 9V, 4 and 9N, 4 and 10A, 4 and 10B, 4 and 11A, 4 and 12F, 4 and 14, 4and 15A, 4 and 15B, 4 and 15C, 4 and 16F, 4 and 17F, 4 and 18C, 4 and 19A, 4 and 19F, 4 and 20, 4 and 20B, 4 and 21, 4 and 22A, 4 and 22F, 4 and 23A, 4 and 23B, 4 and 23F, 4 and 24B, 4 and 24F, 4 and 27, 4 and 29, 4 and 31, 4 and 33B, 4 and 33F, 4 and 34, 4 and 35B, 4 and 35F, 4 and 38, 4 and 72, or 4 and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least athird, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 5, 6A, 6B, 6C, 7C, 7F,8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In some embodiments, the disclosure provides a purified preparation of any of the foregoing glycoconjugates in which the stoichiometric mass ratio of the first and second, first and third, and / or In any of the foregoing embodiments, any two of theazido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in amass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value betweenor range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 5, and the atleast second serotype is selected from the group consisting of serotypes 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 5 and 6A, 5 and 6B, 5 and 6C, 5 and 7C, 5 and 7F, 5 and 8, 5 and 9V, 5 and 9N, 5 and 10A, 5 and 10B, 5 and 11A, 5 and 12F, 5 and 14, 5 and 15A, 5 and 15B, 5 and 15C, 5 and 16F, 5 and 17F, 5 and 18C, 5 and 19A, 5 and 19F, 5 and 20, 5 and 20B, 5 and 21, 5 and 22A, 5 and 22F, 5 and 23A, 5 and 23B, 5 and 23F, 5 and 24B, 5 and 24F, 5 and 27, 5 and 29, 5 and 31, 5 and 33B, 5 and 33F, 5 and 34, 5 and 35B, 5 and 35F, 5 and 38, 5 and 72, or 5 and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third,fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharideis of a distinct S. pneumoniae serotype from each other and from each of said lower order numberedserotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N,10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two ofthe azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixturein a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 6A, and the atleast second serotype is selected from the group consisting of serotypes 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 6A and 6B, 6A and 6C, 6A and 7C, 6A and 7F, 6A and 8, 6A and 9V, 6A and 9N, 6A and 10A, 6A and 10B, 6A and 11A, 6A and 12F, 6A and 14, 6A and 15A, 6A and 15B, 6A and 15C, 6A and 16F, 6A and 17F, 6A and 18C, 6A and 19A, 6A and 19F, 6A and 20, 6A and 20B, 6A and 21, 6A and 22A, 6A and 22F, 6A and 23A, 6A and 23B, 6A and 23F, 6A and 24B, 6A and 24F, 6A and 27, 6A and 29, 6A and 31, 6A and 33B, 6A and 33F, 6A and 34, 6A and 35B, 6A and 35F, 6A and 38, 6A and 72, or 6A and 73. In some embodiments, the reaction mixture comprises azido activatedcapsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at leastthird, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and fromeach of said lower order numbered serotypes, and is selected from the group consisting of serotypes 1,2, 3, 4, 5, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of theforegoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotypecan be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value>1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 6B, and the atleast second serotype is selected from the group consisting of serotypes 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 6B and 6C, 6B and 7C, 6B and 7F, 6B and 8, 6B and 9V, 6B and 9N, 6B and 10A, 6B and 10B, 6B and 11A, 6B and 12F, 6B and 14, 6B and 15A, 6B and 15B, 6B and 15C, 6B and 16F, 6B and 17F, 6B and 18C, 6B and 19A, 6B and 19F, 6B and 20, 6B and 20B, 6B and 21, 6B and 22A, 6B and 22F, 6B and 23A, 6B and 23B, 6B and 23F, 6B and 24B, 6B and 24F, 6B and 27, 6B and 29, 6B and 31, 6B and 33B, 6B and 33F, 6B and 34, 6B and 35B, 6B and 35F, 6B and 38, 6B and 72, or 6B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides ofat least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth orsixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6C, 7C, 7F, 8,9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 6C, and the atleast second serotype is selected from the group consisting of serotypes 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 6C and 7C, 6C and 7F, 6C and 8, 6C and 9V, 6C and 9N, 6C and 10A, 6C and 10B, 6C and 11A, 6C and 12F, 6C and 14, 6C and 15A, 6C and 15B, 6C and 15C, 6C and 16F, 6C and 17F, 6C and 18C, 6C and 19A, 6C and 19F, 6C and 20, 6C and 20B, 6C and 21, 6C and 22A, 6C and 22F, 6C and 23A, 6C and 23B, 6C and 23F, 6C and 24B, 6C and 24F, 6C and 27, 6C and 29, 6C and 31, 6C and 33B, 6C and 33F, 6C and 34, 6C and 35B, 6C and 35F, 6C and 38, 6C and 72, or 6C and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third,fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharideis of a distinct S. pneumoniae serotype from each other and from each of said lower order numberedserotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 7C, 7F, 8, 9V, 9N,10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two ofthe azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixturein a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value betweenor range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 7C, and the atleast second serotype is selected from the group consisting of serotypes 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 7C and 7F, 7C and 8, 7C and 9V, 7C and 9N, 7C and 10A, 7C and 10B, 7C and 11A, 7C and 12F, 7C and 14, 7C and 15A, 7C and 15B, 7C and 15C, 7C and 16F, 7C and17F, 7C and 18C, 7C and 19A, 7C and 19F, 7C and 20, 7C and 20B, 7C and 21, 7C and 22A, 7C and 22F, 7Cand 23A, 7C and 23B, 7C and 23F, 7C and 24B, 7C and 24F, 7C and 27, 7C and 29, 7C and 31, 7C and 33B, 7C and 33F, 7C and 34, 7C and 35B, 7C and 35F, 7C and 38, 7C and 72, or 7C and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixthS. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F,14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B,33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 7F, and the atleast second serotype is selected from the group consisting of serotypes 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 7F and 8, 7F and 9V, 7F and 9N, 7F and 10A, 7F and 10B, 7F and 11A, 7F and 12F, 7F and 14, 7F and 15A, 7F and 15B, 7F and 15C, 7F and 16F, 7F and 17F, 7F and 18C, 7F and 19A, 7F and 19F, 7F and 20, 7F and 20B, 7F and 21, 7F and 22A, 7F and 22F, 7F and 23A, 7F and 23B, 7F and 23F, 7F and 24B, 7F and 24F, 7F and 27, 7F and 29, 7F and 31, 7F and 33B, 7F and 33F, 7F and 34, 7F and 35B, 7F and 35F, 7F and 38, 7F and 72, or 7F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 8, 9V, 9N, 10A, 10B, 11A, 12F,14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or rangeencompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 8, and the atleast second serotype is selected from the group consisting of serotypes 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 8 and 9V, 8 and 9N, 8 and 10A, 8 and 10B, 8 and 11A, 8 and 12F, 8 and 14, 8 and 15A, 8 and 15B, 8 and 15C, 8 and 16F, 8 and 17F, 8 and 18C, 8 and 19A, 8 and 19F, 8 and 20, 8 and 20B, 8 and 21, 8 and 22A, 8 and 22F, 8 and 23A, 8 and 23B, 8 and 23F, 8 and 24B, 8 and 24F, 8 and 27, 8 and 29, 8 and 31, 8 and 33B, 8 and 33F, 8 and 34, 8 and 35B, 8 and 35F, 8 and 38, 8 and 72, or 8 and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides ofat least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth orsixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from eachother by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 9V, and the atleast second serotype is selected from the group consisting of serotypes 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 9V and 9N, 9V and 10A, 9V and 10B, 9V and 11A, 9V and 12F, 9V and 14, 9V and 15A, 9V and 15B, 9V and 15C, 9V and 16F, 9V and 17F, 9V and 18C, 9V and 19A, 9V and 19F, 9V and 20, 9V and 20B, 9V and 21, 9V and 22A, 9V and 22F, 9V and 23A, 9V and 23B, 9V and 23F, 9V and 24B, 9V and 24F, 9V and 27, 9V and 29, 9V and 31, 9V and 33B, 9V and 33F, 9V and 34, 9V and 35B, 9V and 35F, 9V and 38, 9V and 72, or 9V and 73. In some embodiments, the reaction mixture comprisesazido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype,wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype fromeach other and from each of said lower order numbered serotypes, and is selected from the groupconsisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F,17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S.pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein theantecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0,or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of theforegoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 9N, and theat least second serotype is selected from the group consisting of serotypes 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 9N and 10A, 9N and 10B, 9N and 11A, 9N and 12F, 9N and 14, 9N and 15A, 9N and 15B, 9N and 15C, 9N and 16F, 9N and 17F, 9N and 18C, 9N and 19A, 9N and 19F, 9N and 20, 9N and 20B, 9N and 21, 9N and 22A, 9N and 22F, 9N and 23A, 9N and 23B, 9N and 23F, 9N and 24B, 9N and 24F, 9N and 27, 9N and 29, 9N and 31, 9N and 33B, 9N and 33F, 9N and 34, 9N and 35B, 9N and 35F, 9N and 38, 9N and 72, or 9N and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A,19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specificallyenumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 10A, and theat least second serotype is selected from the group consisting of serotypes 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 10A and 10B, 10A and 11A, 10A and 12F, 10A and 14, 10A and 15A, 10A and 15B, 10A and 15C, 10A and 16F, 10A and 17F, 10A and 18C, 10A and 19A, 10A and 19F, 10A and 20, 10A and 20B, 10A and 21, 10A and 22A, 10A and 22F, 10A and 23A, 10A and 23B, 10A and 23F, 10A and 24B, 10A and 24F, 10A and 27, 10A and 29, 10A and 31, 10A and 33B, 10A and 33F, 10A and 34, 10A and 35B, 10A and 35F, 10A and 38, 10A and 72, or 10A and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower order numbered serotypes, and is selected fromthe group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10B, 11A, 12F, 14, 15A, 15B,15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides ofdistinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, whereinthe antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio ofabout 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 10B, and theat least second serotype is selected from the group consisting of serotypes 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 10B and 11A, 10B and 12F, 10B and 14, 10B and 15A, 10B and 15B, 10B and 15C, 10B and 16F, 10B and 17F, 10B and 18C, 10B and 19A, 10B and 19F, 10B and 20, 10B and 20B, 10B and 21, 10B and 22A, 10B and 22F, 10B and 23A, 10B and 23B, 10B and 23F, 10B and 24B, 10B and 24F, 10B and 27, 10B and 29, 10B and 31, 10B and 33B, 10B and 33F, 10B and 34, 10B and 35B, 10B and 35F, 10B and 38, 10B and 72, or 10B and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A,19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 11A, and theat least second serotype is selected from the group consisting of serotypes 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 11A and 12F, 11A and 14, 11A and 15A, 11A and 15B, 11A and 15C, 11A and 16F, 11A and 17F, 11A and 18C, 11A and 19A, 11A and 19F, 11A and 20, 11A and 20B, 11A and 21, 11A and 22A, 11A and 22F, 11A and 23A, 11A and 23B, 11A and 23F, 11A and 24B, 11A and 24F, 11A and 27, 11A and 29, 11A and 31, 11A and 33B, 11A and 33F, 11A and 34, 11A and 35B, 11A and 35F, 11A and 38, 11A and 72, or 11A and 73. In some embodiments, the reaction mixture comprises azido activated capsularsaccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third,fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each ofsaid lower order numbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5,6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A,22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoingembodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less,or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for exampleat least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 12F, and theat least second serotype is selected from the group consisting of serotypes 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 12F and 14, 12F and 15A, 12F and 15B, 12F and 15C, 12F and 16F, 12F and 17F, 12F and 18C, 12F and 19A, 12F and 19F, 12F and 20, 12F and 20B, 12F and 21, 12F and 22A, 12F and 22F, 12F and 23A, 12F and 23B, 12F and 23F, 12F and 24B, 12F and 24F, 12F and 27, 12F and 29, 12F and 31, 12F and 33B, 12F and 33F, 12F and 34, 12F and 35B, 12F and 35F, 12F and 38, 12F and 72, or 12F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least athird, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or anyvalue between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 14, and the atleast second serotype is selected from the group consisting of serotypes 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 14 and 15A, 14 and 15B, 14 and 15C, 14 and 16F, 14 and 17F, 14 and 18C, 14 and 19A, 14 and 19F, 14 and 20, 14 and 20B, 14 and 21, 14 and 22A, 14 and 22F, 14 and 23A, 14 and 23B, 14 and 23F, 14 and 24B, 14 and 24F, 14 and 27, 14 and 29, 14 and 31, 14 and 33B, 14 and 33F, 14 and 34, 14 and 35B, 14 and 35F, 14 and 38, 14 and 72, or 14 and 73. In some embodiments, the reaction mixture comprisesazido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype,wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype fromeach other and from each of said lower order numbered serotypes, and is selected from the groupconsisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 15A, 15B, 15C, 16F,17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S.pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein theantecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0,or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 15A, and theat least second serotype is selected from the group consisting of serotypes 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 15A and 15B, 15A and 15C, 15A and 16F, 15A and 17F, 15A and 18C, 15A and 19A, 15A and 19F, 15A and 20, 15A and 20B, 15A and 21, 15A and 22A, 15A and 22F, 15A and 23A, 15A and 23B, 15A and 23F, 15A and 24B, 15A and 24F, 15A and 27, 15A and 29, 15A and 31, 15A and 33B, 15A and 33F, 15A and 34, 15A and 35B, 15A and 35F, 15A and 38, 15A and 72, or 15A and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 15B, and theat least second serotype is selected from the group consisting of serotypes 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 15B and 15C, 15B and 16F, 15B and 17F, 15B and 18C, 15B and 19A, 15B and 19F, 15B and 20, 15B and 20B, 15B and 21, 15B and 22A, 15B and 22F, 15B and 23A, 15B and 23B, 15B and 23F, 15B and 24B, 15B and 24F, 15B and 27, 15B and 29, 15B and 31, 15B and 33B, 15B and 33F, 15B and 34, 15B and 35B, 15B and 35F, 15B and 38, 15B and 72, or 15B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower order numbered serotypes, and is selected fromthe group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A,15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides ofdistinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, whereinthe antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio ofabout 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 15C, and theat least second serotype is selected from the group consisting of serotypes 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 15C and 16F, 15C and 17F, 15C and 18C, 15C and 19A, 15C and 19F, 15C and 20, 15C and 20B, 15C and 21, 15C and 22A, 15C and 22F, 15C and 23A, 15C and 23B, 15C and 23F, 15C and 24B, 15C and 24F, 15C and 27, 15C and 29, 15C and 31, 15C and 33B, 15C and 33F, 15C and 34, 15C and 35B, 15C and 35F, 15C and 38, 15C and 72, or 15C and 73. In some embodiments, the reaction mixture comprises azido activatedcapsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at leastthird, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and fromeach of said lower order numbered serotypes, and is selected from the group consisting of serotypes 1,2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of theforegoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotypecan be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value>1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 16F, and theat least second serotype is selected from the group consisting of serotypes 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 16F and 17F, 16F and 18C, 16F and 19A, 16F and 19F, 16F and 20, 16F and 20B, 16F and 21, 16F and 22A, 16F and 22F, 16F and 23A, 16F and 23B, 16F and 23F, 16F and 24B, 16F and 24F, 16F and 27, 16F and 29, 16F and 31, 16F and 33B, 16F and 33F, 16F and 34, 16F and 35B, 16F and 35F, 16F and 38, 16F and 72, or 16F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 17F, and theat least second serotype is selected from the group consisting of serotypes 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 17F and 18C, 17F and 19A, 17F and 19F, 17F and 20, 17F and 20B, 17F and 21, 17F and 22A, 17F and 22F, 17F and 23A, 17F and 23B, 17F and 23F, 17F and 24B, 17F and 24F, 17F and 27, 17F and 29, 17F and 31, 17F and 33B, 17F and 33F, 17F and 34, 17F and 35B, 17F and 35F, 17F and 38, 17F and 72, or 17F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third,fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharideis of a distinct S. pneumoniae serotype from each other and from each of said lower order numberedserotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N,10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of theazido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in amass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value betweenor range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 18C, and theat least second serotype is selected from the group consisting of serotypes 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 18C and 19A, 18C and 19F, 18C and 20, 18C and 20B, 18C and 21, 18C and 22A, 18C and 22F, 18C and 23A, 18C and 23B, 18C and 23F, 18C and 24B, 18C and 24F, 18C and 27, 18C and 29, 18C and 31, 18C and 33B, 18C and 33F, 18C and 34, 18C and 35B, 18C and 35F, 18C and 38, 18C and 72, or 18C and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixthS. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or rangeencompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 19A, and theat least second serotype is selected from the group consisting of serotypes 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 19A and 19F, 19Aand 20, 19A and 20B, 19A and 21, 19A and 22A, 19A and 22F, 19A and 23A, 19A and 23B, 19A and 23F,19A and 24B, 19A and 24F, 19A and 27, 19A and 29, 19A and 31, 19A and 33B, 19A and 33F, 19A and 34, 19A and 35B, 19A and 35F, 19A and 38, 19A and 72, or 19A and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 19F, and theat least second serotype is selected from the group consisting of serotypes 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 19F and 20, 19F and 20B, 19F and 21, 19F and 22A, 19F and 22F, 19F and 23A, 19F and 23B, 19F and 23F, 19F and 24B, 19F and 24F, 19F and 27, 19F and 29, 19F and 31, 19F and 33B, 19F and 33F, 19F and 34, 19F and 35B, 19F and 35F, 19F and 38, 19F and 72, or 19F and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C,19A, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 20, and the atleast second serotype is selected from the group consisting of serotypes 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 20 and 20B, 20 and 21, 20 and 22A, 20 and 22F, 20 and 23A, 20 and 23B, 20 and 23F, 20 and 24B, 20 and 24F, 20 and 27, 20 and 29, 20and 31, 20 and 33B, 20 and 33F, 20 and 34, 20 and 35B, 20 and 35F, 20 and 38, 20 and 72, or 20 and 73.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least athird, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 20B, and theat least second serotype is selected from the group consisting of serotypes 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 20B and 21, 20B and 22A, 20Band 22F, 20B and 23A, 20B and 23B, 20B and 23F, 20B and 24B, 20B and 24F, 20B and 27, 20B and 29, 20Band 31, 20B and 33B, 20B and 33F, 20B and 34, 20B and 35B, 20B and 35F, 20B and 38, 20B and 72, or 20B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 21, and the atleast second serotype is selected from the group consisting of serotypes 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 21 and 22A, 21 and 22F, 21 and 23A, 21 and 23B, 21 and 23F, 21 and 24B, 21 and 24F, 21 and 27, 21 and 29, 21 and 31, 21 and 33B, 21 and 33F,21 and 34, 21 and 35B, 21 and 35F, 21 and 38, 21 and 72, or 21 and 73. In some embodiments, the reactionmixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 22A, and theat least second serotype is selected from the group consisting of serotypes 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 22A and 22F, 22A and 23A, 22A and 23B, 22A and 23F, 22A and 24B, 22A and 24F, 22A and 27, 22A and 29, 22A and 31, 22A and 33B, 22A and 33F, 22A and 34, 22A and 35B, 22A and 35F, 22A and 38, 22A and 72, or 22A and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixthS. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or rangeencompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 22F, and theat least second serotype is selected from the group consisting of serotypes 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 22F and 23A, 22F and 23B, 22F and 23F, 22F and 24B, 22F and 24F, 22F and 27, 22F and 29, 22F and 31, 22F and 33B, 22F and 33F, 22F and 34, 22F and 35B, 22F and 35F, 22F and 38, 22F and 72, or 22F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower order numbered serotypes, and is selected fromthe group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A,15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides ofdistinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, whereinthe antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio ofabout 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of theforegoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 23A, and theat least second serotype is selected from the group consisting of serotypes 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 23A and 23B, 23A and 23F, 23A and 24B, 23A and 24F,23A and 27, 23A and 29, 23A and 31, 23A and 33B, 23A and 33F, 23A and 34, 23A and 35B, 23A and 35F,23A and 38, 23A and 72, or 23A and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C,19A, 19F, 20, 20B, 21, 22A, 22F, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 23B, and theat least second serotype is selected from the group consisting of serotypes 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 23B and 23F, 23B and 24B, 23B and 24F, 23B and 27, 23Band 29, 23B and 31, 23B and 33B, 23B and 33F, 23B and 34, 23B and 35B, 23B and 35F, 23B and 38, 23Band 72, or 23B and 73. In some embodiments, the reaction mixture comprises azido activated capsularsaccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third,fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each ofsaid lower order numbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5,6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21,22A, 22F, 23A, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoingembodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be addedto the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less,or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for exampleat least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, forexample, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 23F, and theat least second serotype is selected from the group consisting of serotypes 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 23F and 24B, 23F and 24F, 23F and 27, 23F and 29, 23F and 31, 23F and 33B, 23F and 33F, 23F and 34, 23F and 35B, 23F and 35F, 23F and 38, 23F and 72, or 23F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least athird, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 24B, and theat least second serotype is selected from the group consisting of serotypes 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 24B and 24F, 24B and 27, 24B and 29, 24B and 31, 24B and 33B, 24B and 33F, 24B and 34, 24B and 35B, 24B and 35F, 24B and 38, 24B and 72, or 24B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third,fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharideis of a distinct S. pneumoniae serotype from each other and from each of said lower order numberedserotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N,10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of theazido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in amass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 24F, and theat least second serotype is selected from the group consisting of serotypes 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 24F and 27, 24F and 29, 24F and 31, 24F and 33B, 24F and 33F, 24F and34, 24F and 35B, 24F and 35F, 24F and 38, 24F and 72, or 24F and 73. In some embodiments, the reactionmixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or rangeencompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 27, and the atleast second serotype is selected from the group consisting of serotypes 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 27 and 29, 27 and 31, 27 and 33B, 27 and 33F, 27 and 34, 27 and 35B, 27 and 35F, 27 and 38, 27 and 72, or 27 and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C,19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 29, and the atleast second serotype is selected from the group consisting of serotypes 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 29 and 31, 29 and 33B, 29 and 33F, 29 and 34, 29 and 35B, 29 and 35F, 29 and 38, 29 and 72, or 29 and 73. In some embodiments, the reaction mixture comprises azido activatedcapsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at leastthird, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and fromeach of said lower order numbered serotypes, and is selected from the group consisting of serotypes 1,2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of theforegoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotypecan be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value>1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specificallyenumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 31, and the at least second serotype is selected from the group consisting of serotypes 33B, 33F, 34, 35B, 35F, 38, 72, and 73, for example, 31 and 33B, 31 and 33F, 31 and 34, 31 and 35B, 31 and 35F, 31 and 38, 31 and 72, or 31 and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides ofat least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth orsixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 33B, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example,>1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 33B, and theat least second serotype is selected from the group consisting of serotypes 33F, 34, 35B, 35F, 38, 72, and 73, for example, 33B and 33F, 33B and 34, 33B and 35B, 33B and 35F, 33B and 38, 33B and 72, or 33B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at leasta third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixthsaccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower ordernumbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C,7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A,23B, 23F, 24B, 24F, 27, 29, 31, 33F, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments,any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reactionmixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can beadded to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at leastor about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or anyvalue between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 33F, and theat least second serotype is selected from the group consisting of serotypes 34, 35B, 35F, 38, 72, and 73, for example, 33F and 34, 33F and 35B, 33F and 35F, 33F and 38, 33F and 72, or 33F and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third,fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharideis of a distinct S. pneumoniae serotype from each other and from each of said lower order numberedserotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N,10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 34, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of theazido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in amass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by notmore than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to thereaction mixture in a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 34, and the atleast second serotype is selected from the group consisting of serotypes 35B, 35F, 38, 72, and 73, for example, 34 and 35B, 34 and 35F, 34 and 38, 34 and 72, or 34 and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S.pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S.pneumoniae serotype from each other and from each of said lower order numbered serotypes, and isselected from the group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A,12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 35B, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activatedsaccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio ofabout 1:1, wherein the antecedent and consequent values differ from each other by not more than about±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixturein a mass ratio of about 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or rangeencompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 35B, and theat least second serotype is selected from the group consisting of serotypes 35F, 38, 72, and 73, for example, 35B and 35F, 35B and 38, 35B and 72, or 35B and 73. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each of said lower order numbered serotypes, and is selected fromthe group consisting of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A,15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35F, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides ofdistinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, whereinthe antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio ofabout 1:n, where n is any value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of theforegoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2.In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 35F, and theat least second serotype is selected from the group consisting of serotypes 38, 72, and 73, for example, 35F and 38, 35F and 72, or 35F and 73. In some embodiments, the reaction mixture comprises azidoactivated capsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, whereinsaid at least third, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each otherand from each of said lower order numbered serotypes, and is selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C,19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 38, 72, and 73. In any of the foregoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n isany value >1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 38, and the atleast second serotype is selected from the group consisting of serotypes 72, and 73, for example, 38 and 72, or 38 and 73. In some embodiments, the reaction mixture comprises azido activated capsularsaccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at least third,fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and from each ofsaid lower order numbered serotypes, and is selected from the group consisting of serotypes 1, 2, 3, 4, 5,6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 72, and 73. In any of the foregoingembodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotype can be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less,or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value >1, for exampleat least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. In some embodiments, the reaction mixture comprises azido activated capsular saccharides of atleast 2 distinct S. pneumoniae bacterial serotypes wherein one of the serotypes is serotype 72 and the atleast second serotype is 73. In some embodiments, the reaction mixture comprises azido activatedcapsular saccharides of at least a third, fourth, fifth or sixth S. pneumoniae serotype, wherein said at leastthird, fourth, fifth or sixth saccharide is of a distinct S. pneumoniae serotype from each other and fromeach of said lower order numbered serotypes, and is selected from the group consisting of serotypes 1,2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, and 38. In any of theforegoing embodiments, any two of the azido activated saccharides of distinct S. pneumoniae serotypecan be added to the reaction mixture in a mass ratio of about 1:1, wherein the antecedent and consequent values differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,or 5% or less, or can be added to the reaction mixture in a mass ratio of about 1:n, where n is any value>1, for example at least or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, or higher values, such as 3, 4, 5, or more, or any value between or range encompassing any of the foregoing specifically enumerated values, for example, >1 to 5, or >1 to 4, or >1 to 3, or >1 to 2. Mass ratio of azido activated saccharide to alkyne functionalized carrier proteinIn some embodiments the ratio of the total mass of all azido activated saccharides added to thereaction mixture relative to the total mass of alkyne functionalized carrier protein added to the reactionmixture (defined as the initial input ratio) is between 0.1 and 3 on a weight by weight (w / w) basis. In someembodiments the initial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrierprotein at is between 0.5 and 2. In some embodiments the initial input ratio (w / w) of azido activatedsaccharide to alkyne functionalized carrier protein at is between 0.6 and 1.5. In some embodiments theinitial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at is between0.8 and 1. In some embodiments the initial input ratio (w / w) of azido activated saccharide to alkynefunctionalized carrier protein at is about 0.5. In some embodiments the initial input ratio (w / w) of azidoactivated saccharide to alkyne functionalized carrier protein at is about 0.6. In some embodiments theinitial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at is about0.7. In some embodiments the initial input ratio (w / w) of azido activated saccharide to alkynefunctionalized carrier protein at is about 0.8. In some embodiments the initial input ratio (w / w) of azidoactivated saccharide to alkyne functionalized carrier protein at is about 0.9. In some embodiments theinitial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at is about1. In some embodiments the initial input ratio (w / w) of azido activated saccharide to alkyne functionalizedcarrier protein at is about 1.1. In some embodiments the initial input ratio (w / w) of azido activatedsaccharide to alkyne functionalized carrier protein at is about 1.2. In some embodiments the initial inputratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at is about 1.3. In someembodiments the initial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrierprotein at is about 1.4. In some embodiments the initial input ratio (w / w) of azido activated saccharide toalkyne functionalized carrier protein at is about 1.5. In some embodiments the initial input ratio (w / w) ofazido activated saccharide to alkyne functionalized carrier protein at is about 1.6. In some embodimentsthe initial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at isabout 1.7. In some embodiments the initial input ratio (w / w) of azido activated saccharide to alkynefunctionalized carrier protein at is about 1.8. In some embodiments the initial input ratio (w / w) of azidoactivated saccharide to alkyne functionalized carrier protein at is about 1.9. In some embodiments theinitial input ratio (w / w) of azido activated saccharide to alkyne functionalized carrier protein at is about2. Capping unreacted azido groups after cycloaddition reactionIn some embodiments, after the conjugation reaction, any unreacted azido groups present in theglycoconjugates may be capped using a suitable azido group capping agent. Accordingly, in someembodiments, methods of the disclosure for producing a capsular saccharide glycoconjugate furthercomprise the step of capping unreacted azido groups in the glycoconjugates. In some embodiments, thisstep is accomplished by adding an azido group capping agent to the conjugation reaction mixture in whichthe glycoconjugates were formed. In some embodiments an azido group capping agent is an agent bearing an alkyne group, including terminal alkynes or cycloalkynes. In some embodiments, an azido group capping agent is a compound of formula (V):(V),wherein X is (CH2)n wherein n is selected from 1 to 15.In some embodiments an azido group capping agent is propargyl alcohol.In some embodiments the capping of the unreacted azido groups is performed with an amountof capping agent that is between 0.05 to 20 molar equivalents to the amount of polysaccharide repeatunit of the activated saccharide used in the conjugation reaction. In some embodiments the capping ofthe unreacted azido groups is performed with an amount of capping agent that is between 0.1 to 15 molarequivalents to the amount of polysaccharide repeat unit of the activated saccharide used in theconjugation reaction. In some embodiments the capping of the unreacted azido groups is performed withan amount of capping agent that is between 0.5 to 10 molar equivalents to the amount of polysacchariderepeat unit of the activated saccharide used in the conjugation reaction. In some embodiments thecapping of the unreacted azido groups is performed with an amount of capping agent that is between 0.5to 5 molar equivalents to the amount of polysaccharide repeat unit of the activated saccharide used inthe conjugation reaction. In some embodiments the capping of the unreacted azido groups is performedwith an amount of capping agent that is between 0.5 to 2 molar equivalents to the amount ofpolysaccharide repeat unit of the activated saccharide used in the conjugation reaction. In someembodiments the capping of the unreacted azido groups is performed with an amount of capping agentthat is between 0.5 to 1 molar equivalents to the amount of polysaccharide repeat unit of the activatedsaccharide used in the conjugation reaction. In some embodiments the capping of the unreacted azidogroups is performed with an amount of capping agent that is between 1 to 2 molar equivalents to theamount of polysaccharide repeat unit of the activated saccharide used in the conjugation reaction. Insome embodiments the capping of the unreacted azido groups is performed with an amount of cappingagent that is between 0.75 to 1.5 molar equivalents to the amount of polysaccharide repeat unit of theactivated saccharide used in the conjugation reaction. In some embodiments the capping of the unreactedazido groups is performed with an amount of capping agent that is about 1 molar equivalents to thenumber of polysaccharide repeat units of the activated saccharide used in the conjugation reaction. Insome embodiments the capping of the unreacted azido groups is performed with an amount of cappingagent that is about 1.5 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide used in the conjugation reaction. In some embodiments the capping of the unreacted azidogroups is performed with an amount of capping agent that is about 0.5 molar equivalents to the numberof polysaccharide repeat units of the activated saccharide used in the conjugation reaction. In someembodiments the capping of the unreacted azido groups is performed with an amount of capping agentthat is about 2 molar equivalents to the amount of polysaccharide repeat unit of the activated saccharide used in the conjugation reaction. Capping unreacted alkyne groups after cycloaddition reactionIn some embodiments, after the conjugation reaction, any unreacted alkyne groups present in theglycoconjugates may be capped using a suitable alkyne group capping agent. Accordingly, in some embodiments, methods of the disclosure for producing a capsular saccharide glycoconjugate furthercomprise the step of capping unreacted alkyne groups in the glycoconjugates. In some embodiments, thisstep is accomplished by adding an alkyne group capping agent to the conjugation reaction mixture in which the glycoconjugates were formed. In some embodiments an alkyne group capping agent is an agent bearing an azido group.In some embodiments, an alkyne group capping agent is a compound of formula (VI):(VI), wherein X is (CH2)n wherein n is selected from 1 to 15.In some embodiments an alkyne group capping agent is 3-azido-1-propanol.In some embodiments the capping of the unreacted alkyne groups is performed with an amountof capping agent that is between 0.05 to 20 molar equivalents to the amount of polysaccharide repeatunit of the activated saccharide used in the conjugation reaction. In some embodiments the capping ofthe unreacted alkyne groups is performed with an amount of capping agent that is between 0.1 to 15molar equivalents to the amount of polysaccharide repeat unit of the activated saccharide used in theconjugation reaction. In some embodiments the capping of the unreacted alkyne groups is performedwith an amount of capping agent that is between 0.5 to 10 molar equivalents to the amount ofpolysaccharide repeat unit of the activated saccharide used in the conjugation reaction. In someembodiments the capping of the unreacted alkyne groups is performed with an amount of capping agentthat is between 0.5 to 5 molar equivalents to the amount of polysaccharide repeat unit of the activatedsaccharide used in the conjugation reaction. In some embodiments the capping of the unreacted alkynegroups is performed with an amount of capping agent that is between 0.5 to 2 molar equivalents to theamount of polysaccharide repeat unit of the activated saccharide used in the conjugation reaction. Insome embodiments the capping of the unreacted alkyne groups is performed with an amount of cappingagent that is between 0.5 to 1 molar equivalents to the amount of polysaccharide repeat unit of theactivated saccharide used in the conjugation reaction. In some embodiments the capping of the unreactedalkyne groups is performed with an amount of capping agent that is between 1 to 5 molar equivalents tothe amount of polysaccharide repeat unit of the activated saccharide used in the conjugation reaction. Insome embodiments the capping of the unreacted alkyne groups is performed with an amount of cappingagent that is between 1 to 2 molar equivalents to the amount of polysaccharide repeat unit of theactivated saccharide used in the conjugation reaction. In some embodiments the capping of the unreactedalkyne groups is performed with an amount of capping agent that is between 1.5 to 2.5 molar equivalentsto the amount of polysaccharide repeat unit of the activated saccharide used in the conjugation reaction.In some embodiments the capping of the unreacted alkyne groups is performed with an amount ofcapping agent that about 0.5 molar equivalents to the number of polysaccharide repeat units of theactivated saccharide used in the conjugation reaction. In some embodiments the capping of the unreactedalkyne groups is performed with an amount of capping agent that about 1 molar equivalents to thenumber of polysaccharide repeat units of the activated saccharide used in the conjugation reaction. Insome embodiments the capping of the unreacted alkyne groups is performed with an amount of cappingagent that about 1.5 molar equivalents to the number of polysaccharide repeat units of the activatedsaccharide used in the conjugation reaction. In some embodiments the capping of the unreacted alkynegroups is performed with an amount of capping agent that about 2 molar equivalentS to the amount ofpolysaccharide repeat unit of the activated saccharide used in the conjugation reaction. In someembodiments the capping of the unreacted alkyne groups is performed with an amount of capping agentthat about 2.5 molar equivalents to the amount of polysaccharide repeat unit of the activated saccharideused in the conjugation reaction. In some embodiments the capping of the unreacted alkyne groups isperformed with an amount of capping agent that about 5 molar equivalents to the amount ofpolysaccharide repeat unit of the activated saccharide used in the conjugation reaction.Glycoconjugate purification after cycloaddition reactionIn some embodiments, after the conjugation reaction, glycoconjugates can be purified, partiallyor fully, by a variety of techniques known to the skilled person. These techniques include, withoutlimitation, dialysis, concentration / diafiltration operations against various buffers, tangential flowfiltration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography, others being possible), membrane filtration, and depth filtration. Accordingly, in some embodiments, methods of the disclosure for producing a capsular saccharide glycoconjugate furthercomprise the step of purifying the glycoconjugate, partially or fully, after it is produced.Glycoconjugates produced by click chemistry Covalent linkage between capsular saccharide and carrier protein In some embodiments, the azide-alkyne cycloaddition reactions described herein produce a capsular saccharide glycoconjugate comprising a carrier protein (CP) covalently conjugated to capsularsaccharides of at least two distinct bacterial serotypes through a spacer comprising a 1,4-disubstitutedtriazole, and having the general formula (VII): wherein X is selected from the group consisting of CH2(CH2)n’, (CH2CH2O)mCH2CH2, NHCO(CH2)n’, NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n’ and O(CH2CH2O)mCH2CH2; where n' is selected from 1 to 10 and m is selected from 1 to 4, and wherein X' is selected from the group consisting of CH2O(CH2)n’’CH2C=O, CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4, and where the structure in brackets represents a repeat unit of the capsular saccharide. As will be appreciated, formula (VII) is a schematic representation provided for explication and should not be interpreted as limiting the scope of the inventions. Thus, for example, it should not be inferred that a linkage is necessarily present at every repeat unit of a capsular saccharide (i.e., the structure shown in brackets). Rather, it is possible that a minority of capsular saccharide repeat units arecovalently linked to the carrier protein as illustrated, while the majority of capsular saccharide repeatunits are not linked to the carrier. Additionally, although the schematic shows a single capsular saccharide covalently linked to a single carrier protein, glycoconjugates of the disclosure more typically comprise individual carrier protein (CP) molecules that are covalently linked to a plurality of capsular saccharide molecules of distinct bacterial serotypes. Furthermore, an individual capsular saccharide molecule may be linked to more than one carrier protein (CP) molecule. In some embodiments, the capsular saccharide is not a capsular saccharide from Streptococcuspneumoniae serotype 3.In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is CH2(CH2)n’, where n' is 2 and wherein X' is CH2O(CH2)n’’CH2C=O where n'' is 1. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is CH2(CH2)n’, where n' is selected from 1 to 10 and werein X' is CH2O(CH2)n’’CH2C=O where n'' is selected from 0 to 10. In some embodiments, n' is selected from 1 to 5 and n'' is selected from 0 to 10. In some embodiments, n' is selected from 1 to 5 and n'' is selected from 0 to 5. In some embodiments, n' is selected from 1 to 3 and n'' is selected from 0 to 3. In some embodiments, n' is selected from 1 to 2 and n'' is selected from 0 to 2. In a particular embodiment, n' is 1 and n'' is 0. In another embodiment, n' is 2 and n'' is 0. In yet another embodiment, n' is 3 and n'' is 0. In yet a further embodiment, n' is 4 and n” is 0. In yet a further embodiment, n' is 5 andn” is 0. In yet a further embodiment, n' is 6 and n” is 0. In a particular embodiment, n' is 1 and n'' is 1. Inanother embodiment, n' is 2 and n'' is 1. In yet another embodiment, n' is 3 and n'' is 1. In yet a further embodiment, n' is 4 and n” is 1. In yet a further embodiment, n' is 5 and n” is 1. In yet a further embodiment, n' is 6 and n” is 1. In a particular embodiment, n' is 1 and n'' is 2. In another embodiment, n' is 2 and n'' is 2. In yet another embodiment, n' is 3 and n'' is 2. In yet a further embodiment, n' is 4 and n” is 2. In yet a further embodiment, n' is 5 and n” is 2. In yet a further embodiment, n' is 6 and n” is 2. In a particular embodiment, n' is 1 and n'' is 3. In another embodiment, n' is 2 and n'' is 3. In yet another embodiment, n' is 3 and n'' is 3. In yet a further embodiment, n' is 4 and n” is 3. In yet a further embodiment, n' is 5 and n” is 3. In yet a further embodiment, n' is 6 and n” is 3. In a particular embodiment, n' is 1 and n'' is 4. In another embodiment, n' is 2 and n'' is 4. In yet another embodiment, n' is 3 and n'' is 4. In yet a further embodiment, n' is 4 and n” is 4. In yet a further embodiment, n' is 5 and n” is 4. In yet a further embodiment, n' is 6 and n” is 4. In a particular embodiment, n' is 1 and n'' is 5. In another embodiment, n' is 2 and n'' is 5. In yet another embodiment, n' is 3 and n'' is 5. In yet a further embodiment, n' is 4 and n” is 5. In yet a further embodiment, n' is 5 and n” is 5. In yet a further embodiment, n' is 6 and n” is 5. In a particular embodiment, n' is 1 and n'' is 6. In another embodiment, n' is 2 and n'' is 6. In yet another embodiment, n' is 3 and n'' is 6. In yet a further embodiment, n' is 4 and n” is 6. In yet a further embodiment, n' is 5 and n” is 6. In yet a further embodiment, n' is 6 and n” is 6. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is CH2(CH2)n’, where n' is selected from 1 to 10 and werein CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, where n'' is selected from 0 to 10 and m' is selected from 0 to 4. In some embodiments, n’ is selected from 1 to 5, m’ is selected from 0 to 4 and n’’ is selected from 0 to 10. In some embodiments, n’ is selected from 1 to 5, m’ is selected from 0 to 4 and n’’ is selected from 0 to 5. In some embodiments, n’ is selected from 1 to 3, m’ is selected from 0 to 2 and n’’ is selected from 0 to 3. In some embodiments, n’ is selected from 1 to 2, m’ is selected from 0 to 2 and n’’ is selected from 0 to 1. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 0. In another embodiment, n’ is 1, m’ is 1 and n’’ is 0. In another embodiment, n’ is 1, m’ is 2 and n’’ is 0. In another embodiment, n’ is 1, m’ is 3 and n’’ is 0. In another embodiment, n’ is 2, m’ is 0 and n’’ is 0. In another embodiment, n’ is 2, m’ is 1 and n’’ is 0. In another embodiment, n’ is 2, m’ is 2 and n’’ is 0. In another embodiment, n’ is 2, m’ is 3 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 0. In yet another embodiment,n’ is 3, m’ is 3 and n’’ is 0. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 0. In yet a furtherembodiment, n’ is 4, m’ is 1 and n” is 0. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 0. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 0.In yet a further embodiment, n’ is 5, m’ is 2 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 0. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 1 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 1. In another embodiment, n’ is 2, m’ is 0 and n’’ is 1. In another embodiment, n’ is 2, m’ is 1 and n’’ is 1. In another embodiment, n’ is 2, m’ is 2 and n’’ is 1. In another embodiment, n’ is 2, m’ is 3 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 3 and n’’ is 1. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 1. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 1. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 1. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 1. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 1. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 1. In yet a further embodiment, n’ is 5, m’ is 2 and n” is 1. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 1. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 2. In a particular embodiment, n’ is 1, m’ is 1 and n’’ is 2. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 2. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 2. In another embodiment, n’ is 2, m’ is 0 and n’’ is 2. In another embodiment, n’ is 2, m’ is 1 and n’’ is 2. In another embodiment, n’ is 2, m’ is 2 and n’’ is 2. In another embodiment, n’ is 2, m’ is 3 and n’’ is 2. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 2. In yet another embodiment, n’ is 3, m’ is 1 andn’’ is 2. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 2. In yet another embodiment, n’ is 3, m’ is 3and n’’ is 2. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 2. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 2. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 2. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 2. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 2. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 2. In yet a further embodiment, n’ is 5, m’ is 2 and n” is 2. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 2. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 3. In a particular embodiment, n’ is 1, m’ is 1 and n’’ is 3. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 3. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 3. In another embodiment, n’ is 2, m’ is 0 and n’’ is 3. In another embodiment, n’ is 2, m’ is 1 and n’’ is 3. In another embodiment, n’ is 2, m’ is 2 and n’’ is 3. In another embodiment, n’ is 2, m’ is 3 and n’’ is 3. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 3. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 3. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 3. In yet another embodiment, n’ is 3, m’ is 3 and n’’ is 3. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 3. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 3. In yet a further embodiment, n’ is 4,m’ is 2 and n” is 3. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 3. In yet a further embodiment,n’ is 5, m’ is 0 and n” is 3. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 3. In yet a further embodiment, n’ is 5, m’ is 2 and n” is 3. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 3. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 4. In a particular embodiment, n’ is 1, m’ is 1 and n’’ is 4. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 4. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 4. In another embodiment, n’ is 2, m’ is 0 and n’’ is 4. In another embodiment, n’ is 2, m’ is 1 and n’’ is 4. In another embodiment, n’ is 2, m’ is 2 and n’’ is 4. In another embodiment, n’ is 2, m’ is 3 and n’’ is 4. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 4. In yet another embodiment, n’ is 3, m’ is 1 and n’’is 4. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 4. In yet another embodiment, n’ is 3, m’ is 3 andn’’ is 4. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 4. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 4. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 4. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 4. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 4. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 4. In yet a further embodiment, n’ is 5, m’ is 2 and n” is 4. In yet a furtherembodiment, n’ is 5, m’ is 3 and n” is 4. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 5. In a particularembodiment, n’ is 1, m’ is 1 and n’’ is 5. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 5. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 5. In another embodiment, n’ is 2, m’ is 0 and n’’ is 5. In another embodiment, n’ is 2, m’ is 1 and n’’ is 5. In another embodiment, n’ is 2, m’ is 2 and n’’ is 5. In another embodiment, n’ is 2, m’ is 3 and n’’ is 5. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 5. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 5. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 5. In yet another embodiment, n’ is 3, m’ is 3 and n’’ is 5. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 5. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 5. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 5. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 5. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 5. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 5. In yet a further embodiment, n’ is 5, m’ is 2 and n” is 5. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 5. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is (CH2CH2O)mCH2CH2, where m is selected from 1 to 4 and werein X' is CH2O(CH2)n’’CH2C=O, where n’’ is selected from 0 to 10. In some embodiments, m is selected from 1 to 3 and n’’ is selected from 0 to 10. In some embodiments, m is selected from 1 to 3 and n’’ is selected from 0 to 5. In some embodiments, m is selected from 1 to 2 andn’’ is selected from 0 to 3. In some embodiments, m is selected from 1 to 2 and n’’ is selected from 0 to2. In a particular embodiment, m is 1 and n’’ is 0. In another embodiment, m is 2 and n’’ is 0. In yet another embodiment, m is 3 and n’’ is 0. In yet a further embodiment, m is 4 and n” is 0. In a particular embodiment, m is 1 and n’’ is 1. In another embodiment, m is 2 and n’’ is 1. In yet another embodiment,m is 3 and n’’ is 1. In yet a further embodiment, m is 4 and n” is 1. In a particular embodiment, m is 1 andn’’ is 2. In another embodiment, m is 2 and n’’ is 2. In yet another embodiment, m is 3 and n’’ is 2. In yet a further embodiment, m is 4 and n” is 2. In a particular embodiment, m is 1 and n’’ is 3. In another embodiment, m is 2 and n’’ is 3. In yet another embodiment, m is 3 and n’’ is 3. In yet a further embodiment, m is 4 and n” is 3. In a particular embodiment, m is 1 and n’’ is 4. In another embodiment,m is 2 and n’’ is 4. In yet another embodiment, m is 3 and n’’ is 4. In yet a further embodiment, m is 4 andn” is 4. In a particular embodiment, m is 1 and n’’ is 5. In another embodiment, m is 2 and n’’ is 5. In yetanother embodiment, m is 3 and n’’ is 5. In yet a further embodiment, m is 4 and n” is 5. In a particularembodiment, m is 1 and n’’ is 6. In another embodiment, m is 2 and n’’ is 6. In yet another embodiment, m is 3 and n’’ is 6. In yet a further embodiment, m is 4 and n” is 6. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is (CH2CH2O)mCH2CH2, where m is selected from 1 to 4 and werein X' is CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, where n’’ is selected from 0 to 10 and m’ is selected from 0 to 4. In some embodiments, m is selected from 1 to 3, m’ is selected from 0 to 4 and n’’ is selected from 0 to 10. In some embodiments, m is selected from 1 to 2, m’ is selected from 0 to 4 and n’’ is selected from 0 to 5. In some embodiments, m is selected from 1 to 2, m’ is selected from 0 to 2 and n’’ is selected from 0 to 3. In some embodiments, m is selected from 1 to 2, m’ is selected from 0 to 2 and n’’ is selected from 0 to 1. In a particular embodiment, m is 1, m’ is 0 and n’’ is 0. In another embodiment, m is 1, m’ is 1 and n’’ is 0. In another embodiment, m is 1, m’ is 2 and n’’ is 0. In anotherembodiment, m is 1, m’ is 3 and n’’ is 0. In another embodiment, m is 2, m’ is 0 and n’’ is 0. In anotherembodiment, m is 2, m’ is 1 and n’’ is 0. In another embodiment, m is 2, m’ is 2 and n’’ is 0. In another embodiment, m is 2, m’ is 3 and n’’ is 0. In yet another embodiment, m is 3, m’ is 0 and n’’ is 0. In yet another embodiment, m is 3, m’ is 1 and n’’ is 0. In yet another embodiment, m is 3, m’ is 2 and n’’ is 0. In yet another embodiment, m is 3, m’ is 3 and n’’ is 0. In yet a further embodiment, m is 4, m’ is 0 and n” is 0. In yet a further embodiment, m is 4, m’ is 1 and n” is 0. In yet a further embodiment, m is 4, m’ is 2 and n” is 0. In yet a further embodiment, m is 4, m’ is 3 and n” is 0. In a particular embodiment, m is 1, m’ is 0 and n’’ is 1. In a particular embodiment, m is 1, m’ is 1 and n’’ is 1. In a particular embodiment, m is 1, m’ is 2 and n’’ is 1. In a particular embodiment, m is 1, m’ is 3 and n’’ is 1. In another embodiment, m is 2, m’ is 0 and n’’ is 1. In another embodiment, m is 2, m’ is 1 and n’’ is 1. In another embodiment, m is 2, m’ is 2 and n’’ is 1. In another embodiment, m is 2, m’ is 3 and n’’ is 1. In yet another embodiment, m is 3, m’ is 0 and n’’ is 1. In yet another embodiment, m is 3, m’ is 1 and n’’ is 1. In yet another embodiment, m is 3, m’ is 2 and n’’ is 1. In yet another embodiment, m is 3, m’ is 3 and n’’ is 1. In yet a further embodiment, mis 4, m’ is 0 and n” is 1. In yet a further embodiment, m is 4, m’ is 1 and n” is 1. In yet a further embodiment,m is 4, m’ is 2 and n” is 1. In yet a further embodiment, m is 4, m’ is 3 and n” is 1. In a particular embodiment, m is 1, m’ is 0 and n’’ is 2. In a particular embodiment, m is 1, m’ is 1 and n’’ is 2. In a particular embodiment, m is 1, m’ is 2 and n’’ is 2. In a particular embodiment, m is 1, m’ is 3 and n’’ is 2. In another embodiment, m is 2, m’ is 0 and n’’ is 2. In another embodiment, m is 2, m’ is 1 and n’’ is 2. In another embodiment, m is 2, m’ is 2 and n’’ is 2. In another embodiment, m is 2, m’ is 3 and n’’ is 2. In yet another embodiment, m is 3, m’ is 0 and n’’ is 2. In yet another embodiment, m is 3, m’ is 1 and n’’ is 2. In yet another embodiment, m is 3, m’ is 2 and n’’ is 2. In yet another embodiment, m is 3, m’ is 3 and n’’ is 2. In yet a further embodiment, m is 4, m’ is 0 and n” is 2. In yet a further embodiment, m is 4, m’ is 1 and n” is 2. In yet a further embodiment, m is 4, m’ is 2 and n” is 2. In yet a further embodiment, m is 4, m’ is 3 and n” is 2. In a particular embodiment, m is 1, m’ is 0 and n’’ is 3. In a particular embodiment, m is 1, m’ is 1 and n’’ is 3. In a particular embodiment, m is 1, m’ is 2 and n’’ is 3. In a particular embodiment, m is 1, m’ is 3 and n’’ is 3. In another embodiment, m is 2, m’ is 0 and n’’ is 3. In another embodiment, m is 2, m’ is 1 and n’’ is 3. In another embodiment, m is 2, m’ is 2 and n’’ is 3. In another embodiment, m is 2, m’ is 3 and n’’ is 3. In yet another embodiment, m is 3, m’ is 0 and n’’ is 3. In yet another embodiment, m is 3, m’ is 1 and n’’ is 3. In yet another embodiment, m is 3, m’ is 2 and n’’ is 3. In yet another embodiment, m is 3, m’ is 3 and n’’ is 3. In yet a further embodiment, m is 4, m’ is 0 and n” is 3. In yet a further embodiment, m is 4, m’ is 1 and n” is 3. In yet a further embodiment, m is 4, m’ is 2 and n” is 3. In yet a further embodiment, m is 4, m’ is 3 and n” is 3. In a particular embodiment, m is 1, m’ is 0 and n’’ is 4. In a particular embodiment, m is 1, m’ is 1 and n’’ is 4. In a particular embodiment, m is 1, m’ is 2 and n’’ is 4. In a particular embodiment, m is 1, m’ is 3 and n’’ is 4. In another embodiment, m is 2, m’ is 0 and n’’ is 4. In another embodiment, m is 2, m’ is 1 and n’’ is 4. In another embodiment, m is 2, m’ is 2 and n’’ is 4. In another embodiment, m is 2, m’ is 3 and n’’ is 4. In yet another embodiment, m is 3, m’ is 0 and n’’ is 4.In yet another embodiment, m is 3, m’ is 1 and n’’ is 4. In yet another embodiment, m is 3, m’ is 2 and n’’is 4. In yet another embodiment, m is 3, m’ is 3 and n’’ is 4. In yet a further embodiment, m is 4, m’ is 0and n” is 4. In yet a further embodiment, m is 4, m’ is 1 and n” is 4. In yet a further embodiment, m is 4,m’ is 2 and n” is 4. In yet a further embodiment, m is 4, m’ is 3 and n” is 4. In a particular embodiment, m is 1, m’ is 0 and n’’ is 5. In a particular embodiment, m is 1, m’ is 1 and n’’ is 5. In a particular embodiment, m is 1, m’ is 2 and n’’ is 5. In a particular embodiment, m is 1, m’ is 3 and n’’ is 5. In another embodiment, m is 2, m’ is 0 and n’’ is 5. In another embodiment, m is 2, m’ is 1 and n’’ is 5. In another embodiment, mis 2, m’ is 2 and n’’ is 5. In another embodiment, m is 2, m’ is 3 and n’’ is 5. In yet another embodiment, mis 3, m’ is 0 and n’’ is 5. In yet another embodiment, m is 3, m’ is 1 and n’’ is 5. In yet another embodiment, m is 3, m’ is 2 and n’’ is 5. In yet another embodiment, m is 3, m’ is 3 and n’’ is 5. In yet a further embodiment, m is 4, m’ is 0 and n” is 5. In yet a further embodiment, m is 4, m’ is 1 and n” is 5. In yet a further embodiment, m is 4, m’ is 2 and n” is 5. In yet a further embodiment, m is 4, m’ is 3 and n” is 5. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is NHCO(CH2)n’, where n’ is selected from 1 to 10 and werein X' is CH2O(CH2)n’’CH2C=O, where n’’ is selected from 0 to 10. In some embodiments, n’ is selected from 1 to 5 and n’’ is selected from 0 to 10. In some embodiments, n’ is selected from 1 to 5 and n’’ is selected from 0 to 5. In some embodiments, n’ is selected from 1 to 3 andn’’ is selected from 0 to 3. In some embodiments, n’ is selected from 1 to 2 and n’’ is selected from 0 to2. In a particular embodiment, n’ is 1 and n’’ is 0. In another embodiment, n’ is 2 and n’’ is 0. In yet another embodiment, n’ is 3 and n’’ is 0. In yet a further embodiment, n’ is 4 and n” is 0. In yet a further embodiment, n’ is 5 and n” is 0. In yet a further embodiment, n’ is 6 and n” is 0. In a particular embodiment, n’ is 1 and n’’ is 1. In another embodiment, n’ is 2 and n’’ is 1. In yet another embodiment, n’ is 3 and n’’ is 1. In yet a further embodiment, n’ is 4 and n” is 1. In yet a further embodiment, n’ is 5 and n” is 1. In yet a further embodiment, n’ is 6 and n” is 1. In a particular embodiment, n’ is 1 and n’’ is 2. In another embodiment, n’ is 2 and n’’ is 2. In yet another embodiment, n’ is 3 and n’’ is 2. In yet a further embodiment, n’ is 4 and n” is 2. In yet a further embodiment, n’ is 5 and n” is 2. In yet a further embodiment, n’ is 6 and n” is 2. In a particular embodiment, n’ is 1 and n’’ is 3. In another embodiment, n’ is 2 and n’’ is 3. In yet another embodiment, n’ is 3 and n’’ is 3. In yet a further embodiment, n’ is 4 and n” is 3. In yet a further embodiment, n’ is 5 and n” is 3. In yet a further embodiment, n’ is 6 and n” is 3. In a particular embodiment, n’ is 1 and n’’ is 4. In another embodiment, n’ is 2 and n’’ is 4. In yet another embodiment, n’ is 3 and n’’ is 4. In yet a further embodiment, n’ is 4 and n” is 4. In yet a further embodiment, n’ is 5 and n” is 4. In yet a further embodiment, n’ is 6 and n” is 4. In a particularembodiment, n’ is 1 and n’’ is 5. In another embodiment, n’ is 2 and n’’ is 5. In yet another embodiment,n’ is 3 and n’’ is 5. In yet a further embodiment, n’ is 4 and n” is 5. In yet a further embodiment, n’ is 5 and n” is 5. In yet a further embodiment, n’ is 6 and n” is 5. In a particular embodiment, n’ is 1 and n’’ is 6. Inanother embodiment, n’ is 2 and n’’ is 6. In yet another embodiment, n’ is 3 and n’’ is 6. In yet a furtherembodiment, n’ is 4 and n” is 6. In yet a further embodiment, n’ is 5 and n” is 6. In yet a further embodiment, n’ is 6 and n” is 6. In some embodiments, the disclosure provides a capsular saccharide glycoconjugate comprising capsular saccharides of at least two distinct bacterial serotypes covalently conjugated to a carrier protein (CP) through a spacer and having the general formula (VII), wherein X is NHCO(CH2)n’, where n’ is selected from 1 to 10 and werein X' is CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, where n’’ is selected from 0 to 10 and m’ isselected from 0 to 4. In some embodiments, n’ is selected from 1 to 5, m’ is selected from 0 to 4 and n’’is selected from 0 to 10. In some embodiments, n’ is selected from 1 to 5, m’ is selected from 0 to 4 and n’’ is selected from 0 to 5. In some embodiments, n’ is selected from 1 to 3, m’ is selected from 0 to 2 and n’’ is selected from 0 to 3. In some embodiments, n’ is selected from 1 to 2, m’ is selected from 0 to 2 and n’’ is selected from 0 to 1. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 0. In another embodiment, n’ is 1, m’ is 1 and n’’ is 0. In another embodiment, n’ is 1, m’ is 2 and n’’ is 0. In another embodiment, n’ is 1, m’ is 3 and n’’ is 0. In another embodiment, n’ is 2, m’ is 0 and n’’ is 0. In another embodiment, n’ is 2, m’ is 1 and n’’ is 0. In another embodiment, n’ is 2, m’ is 2 and n’’ is 0. In another embodiment, n’ is 2, m’ is 3 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 0. In yet another embodiment, n’ is 3, m’ is 3 and n’’ is 0. In yet a further embodiment, n’ is 4, m’ is 0 and n” is 0. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 0. In yet a further embodiment, n’ is 4, m’ is 2 and n” is 0. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 1 and n” is 0.In yet a further embodiment, n’ is 5, m’ is 2 and n” is 0. In yet a further embodiment, n’ is 5, m’ is 3 and n” is 0. In a particular embodiment, n’ is 1, m’ is 0 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 1 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 2 and n’’ is 1. In a particular embodiment, n’ is 1, m’ is 3 and n’’ is 1. In another embodiment, n’ is 2, m’ is 0 and n’’ is 1. In another embodiment, n’ is 2, m’ is 1 and n’’ is 1. In another embodiment, n’ is 2, m’ is 2 and n’’ is 1. In another embodiment, n’ is 2, m’ is 3 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 0 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 1 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 2 and n’’ is 1. In yet another embodiment, n’ is 3, m’ is 3 and n’’ is 1. In yet a further embodiment, n’ is 4,m’ is 0 and n” is 1. In yet a further embodiment, n’ is 4, m’ is 1 and n” is 1. In yet a further embodiment,n’ is 4, m’ is 2 and n” is 1. In yet a further embodiment, n’ is 4, m’ is 3 and n” is 1. In yet a further embodiment, n’ is 5, m’ is 0 and n” is 1. In yet a further embodiment, n’ is 5, m’...

Claims

What is claimed is:

1. A method of producing a glycoconjugate comprising reacting an alkyne functionalized carrierprotein with a first and at least a second azido activated capsular saccharide of distinct bacterial serotypes in the same azide-alkyne cycloaddition reaction using Cu(I) as a catalyst.

2. The method of claim 1, wherein the first and at least second azido activated saccharides are addedto the reaction in a mass ratio ranging from about 1:1 to about 1:5.

3. The method of claim 2, wherein the first and at least second azido activated saccharides are addedto the reaction in a mass ratio of about 1:2.

4. The method of claim 2, wherein the first and at least second azido activated saccharides are addedto the reaction in a mass ratio of about 1:1.

5. The method of claim 4, wherein the relative masses of the first and at least second azido activatedsaccharides differ from each other by not more than about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less.

6. The method of any one of claims 1 to 5, wherein the reaction comprises azido activated capsularsaccharides of at least 3 distinct bacterial serotypes.

7. The method of any one of claims 1 to 5, wherein the reaction comprises azido activated capsularsaccharides of at least 4 distinct bacterial serotypes.

8. The method of any one of claims 1 to 5, wherein the reaction comprises azido activated capsularsaccharides of at least 5 distinct bacterial serotypes.

9. The method of any one of claims 1 to 8, wherein said alkyne functionalized carrier protein isprepared by reacting a carrier protein with a compound comprising an N-hydroxysuccinimide (NHS)moiety and an alkyne group, wherein said NHS moiety reacts with an amino group of said carrier protein,forming an amide linkage.

10. The method of claim 9, wherein the alkyne of said compound is a terminal alkyne or a cycloalkyne.

11. The method of any one of claims 9 to 10, wherein said compound has the general formula (I):where X is selected from the group consisting of CH2O(CH2)nCH2C=O and CH2O(CH2CH2O)m(CH2)nCH2C=O, n ranges from 0 to 10, and m ranges from 0 to 4.

12. The method of any one of claims 9 to 11, wherein said compound has the formula (II):(II).

13. The method of any one of claims 9 to 12, wherein said carrier protein is reacted with an amountof said compound that is between 0.10 to 10.00 molar equivalents to the amount of lysine in said carrier protein.

14. The method of any one of claims 1 to 13, wherein the degree of functionalization of said alkynefunctionalized carrier protein ranges from 1 to 50.

15. The method of any one of claims 1 to 8, wherein the azido activated capsular saccharides areprepared by reacting a capsular saccharide with a carbonic acid derivative and an azido linker.

16. The method of claim 15, wherein said azido linker has the formula (III):(III), where X is selected from the group consisting of CH2(CH2)n, (CH2CH2O)mCH2CH2, NHCO(CH2)n, NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n, and O(CH2CH2O)mCH2CH2, n ranges from 1 to 10, and m ranges from 1 to 4.

17. The method of any one of claims 15 to 16, wherein said azido linker has the formula (IV):(IV).

18. The method of claim 15, wherein either or both of said steps of reacting with the carbonic acidderivative or azido linker are carried out in an aprotic solvent.

19. The method of any one of claims 15 to 18, wherein said step of reacting with the carbonic acidderivative is carried out in a solution comprising dimethylsulphoxide (DMSO).

20. The method of any one of claims 15 to 19, wherein the carbonic acid derivative is selected fromthe group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyl-di-(1,2,4-triazole) (CDT), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.

21. The method of claim 18, wherein the carbonic acid derivative is CDI and the aprotic solventcomprises 0.1% to 1% (v / v) water.

22. The method of claim 19, wherein the carbonic acid derivative is CDI and the solution comprisingDMSO comprises 0.1% to 1% (v / v) water.

23. The method of claims 15, further comprising, after reacting the capsular saccharide with thecarbonic acid derivative, adding water.

24. The method of claim 23, wherein the total water content of the reaction ranges from about 1%to about 10% (v / v).

25. The method of any one of claims 15 to 24, wherein the reaction with the azido linker is carriedout with an amount of azido linker that is between 0.01 to 10.00 molar equivalents of the amount of saccharide repeat units in the capsular saccharides.

26. The method of any one of claims 15 to 25, wherein the degree of activation of the azidoactivated capsular saccharides ranges from 0.5% to 50%.

27. The method of any one of claims 1 to 26, wherein the weight average molecular weight (Mw) ofthe capsular saccharides ranges from 10 kDa to 2000 kDa.

28. The method of any one of claims 1 to 27, wherein the reaction further comprises a reducingagent.

29. The method of any one of claims 1 to 28, wherein the reaction further comprises tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and aminoguanidine, wherein reducing agent is ascorbate.

30. The method of any one of claims 1 to 29, wherein the reaction comprises azido activatedcapsular saccharides and alkyne functionalized carrier protein in a mass ratio ranging from 0.1 to 3.0 (w / w).

31. The method of any one of claims 1 to 30, further comprising capping unreacted azido groups withan azido group capping agent.

32. The method of claim 31, wherein the azido group capping agent is a compound of formula (V),(V), where X is (CH2)n and where n is selected from 1 to 15.

33. The method of claim 31, wherein the azido group capping agent is propargyl alcohol.

34. The method of any one of claims 31 to 33, wherein capping azido groups is carried out with anamount of the capping agent that is between 0.05 to 20.00 molar equivalents to the amount of saccharide repeat units of the capsular saccharide.

35. The method of any one of claims 1 to 30, further comprising capping unreacted alkyne groupswith an alkyne group capping agent.

36. The method of claim 35, wherein the alkyne group capping agent comprises an azido group.

37. The method of claim 35, wherein the alkyne group capping agent is a compound of formula (VI),(VI),where X is (CH2)nand where n is selected from 1 to 15.

38. The method of claim 35, wherein the alkyne group capping agent is 3-azido-1-propanol.

39. The method of any one of claims 32-35, wherein capping alkyne groups is carried out with anamount of the capping agent that is between 0.05 to 20.00 molar equivalents to the amount of saccharide repeat units of said capsular saccharide or to the amount of lysine in said carrier protein.

40. The method of any one of claims 1 to 39, further comprising purifying the glycoconjugate.

41. The method of any one of claims 1 to 40, wherein the carrier protein is selected from the groupconsisting of: CRM197; diphtheria toxoid (DT); tetanus toxoid (TT); H. influenzae protein D (PD); SCP or anenzymatically inactive fragment thereof; an enzymatically inactive fragment of an SCP comprising at least one substitution mutation D130A, H193A, N295A or S512A; an enzymatically inactive fragment of an SCP comprising at least the substitution mutations D130A and S512A; an enzymatically inactive fragment of SCP comprising the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence at least 95% identical thereto; and an enzymatically inactive fragment of SCP comprising the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence at least 95% identical thereto.

42. The method of any one of claims 1 to 41, wherein the capsular saccharide is from a pathogenicbacteria selected from the group consisting of: Streptococcus, Staphylococcus, Enterococcus, Bacillus,Corynebacterium, Listeria, Erysipelothrix, Clostridium, Haemophilus, Neisseria, and Escherichia.

43. The method of any one of claims 1 to 41, wherein the capsular saccharide is from a pathogenicStreptococcus bacteria selected from the group consisting of: Group B streptococcus (GBS), Group Cstreptococcus, and Group G streptococcus.

44. The method of any one of claims 1 to 41, wherein the capsular saccharide is from a pathogenicGroup B streptococcus (GBS), bacteria selected from the group consisting of: GBS type Ia, Ib, II, III, IV, V, VI, VII, and VIII.

45. The method of any one of claims 1 to 41, wherein the capsular saccharide is from a pathogenicbacteria selected from the group consisting of: Enterococcus faecalis, Escherichia coli, Haemophilusinfluenzae, Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus agalactiae,Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes.

46. The method of any one of claims 1 to 41, wherein at least one of the capsular saccharides is acapsular saccharide from a Streptococcus pneumoniae serotype selected from the group consisting ofserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73.

47. A bacterial capsular saccharide glycoconjugate produced by the method of any one of claims 1 to46.

48. The glycoconjugate of claim 47, wherein each of the first and and at second capsular saccharidesis covalently conjugated to said carrier protein (CP) through a spacer and has the general formula (VII):where X is selected from the group consisting of CH2(CH2)n’, (CH2CH2O)mCH2CH2, NHCO(CH2)n’, NHCO(CH2CH2O)mCH2CH2, OCH2(CH2)n’ and O(CH2CH2O)mCH2CH2, n' ranges from 1 to 10, and m ranges from 1 to 4; and where X' is selected from the group consisting of CH2O(CH2)n’’CH2C=O, CH2O(CH2CH2O)m’(CH2)n’’CH2C=O, n'' ranges from 0 to 10, and m' ranges from 0 to 4.

49. The glycoconjugate of claim 48, where X is CH2(CH2)n’ and n' is 2; and where X' isCH2O(CH2)n’’CH2C=O and n'' is 1.

50. The glycoconjugate of claims 47, wherein each of the first and and at second capsular saccharidesis covalently conjugated to the carrier protein (CP) through a spacer and has the general formula (VIII):(VIII).

51. The glycoconjugate of any one of claims 47 to 50, wherein the weight average molecular weight(Mw) of either of the first or at least second capsular saccharides before conjugation ranges from 10 kDa to 2,000 kDa.

52. The glycoconjugate of any one of claims 47 to 51, wherein the weight average molecular weight(Mw) of the glycoconjugate ranges from 250 kDa to 20,000 kDa.

53. The glycoconjugate of any one of claims 47 to 52, wherein the degree of conjugation of theglycoconjugate ranges from 2 to 15.

54. The glycoconjugate of any one of claims 47 to 53, wherein the mass ratio of total capsularsaccharides to carrier protein in the glycoconjugate ranges from 0.5 to 3.0 on a weight by weight (w / w)basis.

55. The glycoconjugate of any one of claims 47 to 54, wherein the glycoconjugate comprises at leastone covalent linkage between the carrier protein and the capsular saccharides for every 4 to 25 saccharide repeat units.

56. The glycoconjugate of any one of claims 47 to 55, wherein each of the first and at least secondcapsular saccharides is a capsular saccharide from distinct Streptococcus pneumoniae (S. pneumoniae)serotypes selected from the group of serotypes consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31, 33B, 33F, 34, 35B, 35F, 38, 72, and 73.

57. The glycoconjugate of any one of claims 47 to 56, wherein the carrier protein is CRM197.

58. The glycoconjugate of any one of claims 47 to 56, wherein the carrier protein is SCP.

59. The glycoconjugate of any one of claims 56 to 58, wherein the mass ratio of the first and at leastsecond S. pneumoniae saccharide serotypes conjugated to the carrier protein ranges from about 1:1 toabout 1:5.

60. The glycoconjugate of any one of claims 56 to 58, wherein the mass ratio of the first and at leastsecond S. pneumoniae saccharide serotypes conjugated to the carrier protein is about 1:2.

61. The glycoconjugate of any one of claims 56 to 58, wherein the mass ratio of the first and at leastsecond S. pneumoniae saccharide serotypes conjugated to the carrier protein is about 1:1.

62. The glycoconjugate of claim 61, wherein the mass ratio of the first and at least second S.pneumoniae saccharide serotypes conjugated to the carrier protein differ from each other by not morethan about ±50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less.

63. The glycoconjugate of any one of claims 56 to 62, wherein the glycoconjugate comprises at least3 distinct S. pneumoniae saccharide serotypes.

64. The glycoconjugate of any one of claims 56 to 62, wherein the glycoconjugate comprises at least4 distinct S. pneumoniae saccharide serotypes.

65. The glycoconjugate of any one of claims 56 to 62, wherein the glycoconjugate comprises at least5 distinct S. pneumoniae saccharide serotypes.

66. An immunogenic composition comprising the glycoconjugate of any one of claims 56 to 65.

67. The immunogenic composition of claim 66, comprising a first and at least second glycoconjugate,wherein the first glycoconjugate comprises a carrier protein conjugated to at least 2 distinct S.pneumoniae saccharide serotypes and the second glycoconjugate comprises a carrier protein conjugatedto at least one S. pneumoniae saccharide serotype which is different from the saccharide serotypes of thefirst glycoconjugate.

68. The immunogenic composition of claim 67, wherein the first glycoconjugate comprises a carrierprotein conjugated to at least 3 distinct S. pneumoniae saccharide serotypes.

69. The immunogenic composition of claim 67, wherein the first glycoconjugate comprises a carrierprotein conjugated to at least 4 distinct S. pneumoniae saccharide serotypes.

70. The immunogenic composition of claim 67, wherein the first glycoconjugate comprises a carrierprotein conjugated to at least 5 distinct S. pneumoniae saccharide serotypes.

71. A method of preventing a disease or disorder in a subject caused by a bacterial infection,comprising administering a prophylactically effective amount of an immunogenic composition comprising the glycoconjugate of any one of claims 56 to 65 or the immunogenic composition of any one of claims 66 to 70.

72. A method of treating a disease or disorder in a subject caused by a bacterial infection, comprisingadministering a therapeutically effective amount of an immunogenic composition comprising the glycoconjugate of any one of claims 56 to 65 or the immunogenic composition of any one of claims 66 to 70.

73. The method of any one of claims 71 or 72, wherein the bacterial infection is of Streptococcuspneumoniae bacteria.

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