Improved methods for producing glycoconjugates by reductive amination in aprotic solvent

Abstract

Among its various embodiments, the disclosure provides improved methods for producing glycoconjugates of carrier protein, including CRM197, and bacterial capsular saccharide, including those isolated from various Streptococcus pneumoniae serotypes.

Description

[0001] IMPROVED METHODS FOR PRODUCING GLYCOCONJUGATES BY REDUCTIVE AMINATION IN APROTIC SOLVENT

[0002] BACKGROUND

[0003] Certain bacteria of medical and public health importance produce an extracellular capsule, principally composed of saccharides, 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 saccharides 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.

[0004] Existing methods for producing glycoconjugates involve activating capsular saccharides with compounds to create chemical reactive sites and then reacting the activated saccharides with carrier proteins to form covalent linkages between them. A common reaction for this purpose is reductive amination, in which saccharides are activated with an oxidizing agent after which activated saccharide and carrier protein, such as CRM197, are reacted in the presence of a reducing agent. Reductive amination is often carried out in aqueous solution, but it has been discovered that certain saccharides react poorly when water is used as the solvent. Substituting an aprotic solvent, such as DMSO, has sometimes been effective to improve reaction efficiency, but there remain types of saccharide for which DMSO does not work that well or at all. In particular, certain saccharides are observed to be poorly soluble or insoluble in DMSO, either before or after being mixed with carrier protein which had been compounded with buffers and other excipients, lyophilized, and then reconstituted in DMSO. A related challenge has been the low solubility of carrier protein, in particular CRM197, after reconstitution in DMSO from its lyophilized form, which is a separate factor limiting conjugation efficiency.

[0005] Accordingly, there exists a need in the art for methods of conjugating activated bacterial capsular saccharides and carrier proteins by reductive amination in aprotic solvent where the solubility of the activated saccharides as well as carrier protein are increased, thus leading to enhanced conjugation efficiency and yield of glycoconjugates compared to existing methods.

[0006] SUMMARY

[0007] 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).

[0008] EI . A method for producing a glycoconjugate, comprising reacting an activated saccharide and a carrier protein in the presence of a reducing agent in a solution of an aprotic solvent, wherein said solution further comprises potassium ions.

[0009] E2. The method of E1 , wherein said carrier protein is selected from the group consisting of

[0010] DT, TT, fHBP, PD, SCP, and CRM protein. E3. The method of E2, wherein said CRM protein is CRM197.

[0011] E4. The method of any of the preceding embodiments, wherein said saccharide is a bacterial capsular saccharide.

[0012] E5. The method of E4, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria.

[0013] E6. The method of E4-E5, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria of defined serotype.

[0014] E7. The method of E6, wherein said Spn serotype 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, 38, 72, and 73.

[0015] E8. The method of any of the preceding embodiments, wherein said saccharide is sized.

[0016] E9. The method of any of the preceding embodiments, wherein the weight average molecular weight (Mw) of said saccharide is at least or about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, or 2000 kDa, or ranges from about 5 kDa to about 2000 kDa.

[0017] E10. The method of any of the preceding embodiments, wherein said saccharide is activated by reaction with an oxidizing agent.

[0018] E11. The method of E10, wherein said oxidizing agent is periodate.

[0019] E12. The method of E11 , wherein said periodate is metaperiodate, or salts thereof.

[0020] E13. The method of E11 , wherein said periodate is orthoperiodate, or salts thereof.

[0021] E14. The method of E10-E13, wherein after reaction with oxidizing agent, said saccharide is contacted with a quenching agent.

[0022] E15. The method of any of the preceding embodiments, wherein the degree of oxidation of said saccharide is at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, or ranges from about 2 to about 30.

[0023] E16. The method of any of the preceding embodiments, wherein the weight average molecular weight (Mw) of said activated saccharide is at least or about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, or 5000 kDa, or ranges from about 5 kDa to about 5000 kDa.

[0024] E17. The method of any of the preceding embodiments, wherein prior to reacting, said activated saccharide is reconstituted from a lyophilized composition comprising activated saccharide.

[0025] E18. The method of any of the preceding embodiments, wherein prior to reacting, said carrier protein is reconstituted from a lyophilized composition comprising carrier protein, said lyophilized composition comprising carrier protein optionally further comprising said activated saccharide.

[0026] E19. The method of E17-E18, wherein said activated saccharide or carrier protein is reconstituted with an aprotic solvent.

[0027] E20. The method of E19, wherein said aprotic solvent is dimethylsulfoxide (DMSO).

[0028] E21. The method of E20, wherein said DMSO is anhydrous.

[0029] E22. The method of E18-E21, wherein said lyophilized composition comprising carrier protein further comprises a potassium salt.

[0030] E23. The method of E22, wherein said potassium salt is selected from the group consisting of potassium phosphate monobasic, potassium phosphate dibasic, and potassium chloride.

[0031] E24. The method of E22-E23, wherein the molar ratio of the amount of potassium to the amount of carrier protein in the lyophilized composition comprising carrier protein is at least or about 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550: 1 , 500: 1 , 450: 1 , 400: 1 , 350: 1 , 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4: 1 , 3: 1 , 2: 1 , or 1 : 1 , or ranges from about 1000: 1 to about 1:1, or about 120: 1 to about 60: 1 , or about 100: 1 to about 80: 1.

[0032] E25. The method of E22-E24, wherein said lyophilized composition comprising carrier protein comprises no more than trace amounts of sodium.

[0033] E26. The method of E22-E24, wherein the molar ratio of the amount of potassium to the amount of sodium in the lyophilized composition comprising carrier protein is at least or about 1,000,000:1, 500,000:1, 100,000:1, 50,000:1, 10,000:1, 5000:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1:1, or ranges from about 1,000,000:1 to about 0.1:1. E27. The method of E18-E26, wherein said lyophilized composition comprising carrier protein further comprises a non-reducing sugar.

[0034] E28. The method of E27, wherein said non-reducing sugar is sucrose.

[0035] E29. The method of E28, wherein the relative amount of sucrose to carrier protein in said lyophilized composition comprising carrier protein is at least or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,

[0036] 4.0, 4.5, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,

[0037] 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5,

[0038] 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, or 25.0 times greater, or ranges from about 1.0 to about 25.0 times greater.

[0039] E30. The method of E18-E29, wherein after reconstitution the formerly lyophilized composition comprising carrier protein comprises at least or about 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 millimolar (mM) potassium ions, or ranges from about 0.5 mM to about 90 mM potassium ions.

[0040] E31. The method of E18-E30, wherein after reconstitution the formerly lyophilized composition comprising carrier protein comprises at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 grams carrier protein per liter of aprotic solvent, or ranges from about 3 g / L to about 25 g / L, and wherein said carrier protein is fully solubilized upon reconstitution.

[0041] E32. The method of E18-E31 , wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).

[0042] E33. The method of E18-E32, wherein after reconstitution the formerly lyophilized composition comprising carrier protein comprises not more than 25 mM sodium ions.

[0043] E34. The method of E18-E33, wherein after reconstitution the formerly lyophilized composition comprising carrier protein comprises not more than a trace amount of sodium ions.

[0044] E35. The method of E30-E34, wherein after reconstitution the molar ratio of the amount of potassium to the amount of carrier protein in the formerly lyophilized composition comprising carrier protein is at least or about 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550:1 , 500:1 , 450:1 , 400:1 , 350:1 , 300:1 , 250:1 , 200:1 , 150:1 , 140:1 , 130:1 , 125:1 , 120:1 , 115:1 , 110:1 , 105:1 , 100:1 , 95:1 , 90:1 , 85:1 , 80:1 , 75:1 , 70:1 , 65:1 , 60:1 , 55:1 , 50:1 , 45:1 , 40:1 , 35:1 , 30:1 , 25:1 , 20:1 , 15:1 , 10:1 , 5:1 , 4:1 , 3:1 , 2:1 , or 1 :1 , or ranges from about 1000:1 to about 1 :1 , or about 120: 1 to about 60: 1 , or about 100: 1 to about 80: 1 .

[0045] E36. The method of E30-E35, wherein after reconstitution the molar ratio of the amount of potassium to the amount of sodium in the formerly lyophilized composition comprising carrier protein is at least or about 1,000,000:1, 500,000:1, 100,000:1, 50,000:1, 10,000:1, 5000:1, 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550: 1 , 500: 1 , 450: 1 , 400: 1 , 350: 1 , 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or0.1:1 or ranges from about 1 ,000,000: 1 to about 0.1:1.

[0046] E37. The method of E30-E36, wherein said lyophilized composition comprising carrier protein further comprises sucrose, and wherein after reconstitution the relative amount of sucrose to carrier protein in said formerly lyophilized composition is at least or about 1.0, 1.5, 2.0, 2.5, 3.0,

[0047] 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,

[0048] 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0,

[0049] 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, or 25.0 times greater, or ranges from about 1.0 to about 25.0 times greater.

[0050] E38. The method of E18-E37, wherein prior to reconstituting, said lyophilized composition comprising carrier protein is lyophilized from a lyophilization solution comprising said carrier protein.

[0051] E39. The method of E38, wherein said lyophilization solution is an aqueous solution further comprising potassium phosphate buffer.

[0052] E40. The method of E39, wherein the concentration of said potassium phosphate buffer is at least about 2.5 mM, or at least about 5 mM, or ranges from about 5 mM to about 90 mM, or ranges from about 5mM to about 15 mM.

[0053] E41. The method of E39-E40, wherein the pH of said potassium phosphate buffer ranges from about pH 7.0 to about pH 7.8, or from about pH 7.1 to about pH 7.7, or about pH 7.2 to about pH 7.6, or about pH 7.3 to about pH 7.5, or is about pH 7.4.

[0054] E42. The method of E38-E41, wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).

[0055] E43. The method of E38-E42, wherein said CRM protein is CRM197.

[0056] E44. The method of E38-E43, wherein said lyophilization solution comprises at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 millimolar (mM) potassium ions, or ranges from about 0.5 mM to about 90 mM potassium ions.

[0057] E45. The method of E38-E44, wherein said lyophilization solution comprises at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 grams carrier protein per liter of solution, or ranges from about 3 g / L to about 25 g / L. E46. The method of E38-E45, wherein said lyophilization solution comprises not more than 25 mM sodium ions.

[0058] E47. The method of E38-E46, wherein said lyophilization solution comprises not more than a trace amount of sodium ions.

[0059] E48. The method of E38-E47, wherein said lyophilization solution comprises about 5-40 mM potassium ions.

[0060] E49. The method of E38-E48, wherein said lyophilization solution comprises about 5-40 mM potassium ions and not more than a trace amount of sodium ions.

[0061] E50. The method of E38-E45, wherein the molar ratio of the amount of potassium to the amount of carrier protein in said lyophilization solution is at least or about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, or 1:1, or ranges from about 1000:1 to about 1:1, or about 120:1 to about 60:1, or about 100:1 to about 80:1.

[0062] E51. The method of E38-E45, wherein the molar ratio of the amount of potassium to the amount of sodium in said lyophilization solution is at least or about 1,000,000:1, 500,000:1, 100,000:1, 50,000:1, 10,000:1, 5000:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, or 0.1 : 1 , or ranges from about 1 ,000,000: 1 to about 0.1:1.

[0063] E52. The method of E38-E51, wherein said lyophilization solution further comprises a nonreducing sugar.

[0064] E53. The method of E52, wherein said non-reducing sugar is sucrose.

[0065] E54. The method of E53, wherein said lyophilization solution comprises at least or about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, or 15.0% sucrose (w / v), or ranges from about 0.5% to about 15%, or about 3% to about 6%, or about 4% to about 5%.

[0066] E55. The method of E53, wherein the relative amount of sucrose to carrier protein in said lyophilization solution is at least or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, or 25.0 times greater, or ranges from about 1.0 to about 25.0 times greater.

[0067] E56. The method of E38-E42, wherein said lyophilization solution comprises 3-9 g / L of CRM197 in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0068] E57. The method of E38-E42, wherein said lyophilization solution comprises 3-9 g / L of CRM197 in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0069] E58. The method of E38-E42, wherein said lyophilization solution comprises 4-8 g / L of CRM197 in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0070] E59. The method of E38-E42, wherein said lyophilization solution comprises 4-8 g / L of CRM197 in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0071] E60. The method of E38-E42, wherein said lyophilization solution comprises 5-7 g / L of CRM197 in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0072] E61. The method of E38-E42, wherein said lyophilization solution comprises 5-7 g / L of CRM197 in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, not more than 25 mM Na+ions, and a mass of dissolved sucrose at least 2.5 times the amount of CRM197.

[0073] E62. The method of any of the preceding embodiments, wherein said reducing agent is sodium cyanoborohydride.

[0074] E63. The method of any of the preceding embodiments, wherein the ratio of the amounts of activated saccharide and carrier protein that are reacted is at least or about 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3.5:1 , 3:1 , 2:1 , 1.9:1 , 1.8:1 , 1.7:1 , 1.6:1 , 1.5:1 , 1.4:1 , 1.3:1 , 1.2:1 , 1.1 :1 , 1.0:1 , 0.9:1 , 0.8:1 , 0.7:1 , 0.6:1 , 0.5:1 , 0.4:1 , 0.3:1 , 0.2:1 , or 0.1 :1 , or ranges from about 10:1 to about 0.1 :1 , or about 3:1 to about 0.33:1 , or about 2:1 to about 0.5:1 , or about 1.5:1 to about 0.67:1 , or about 1.4:1 to about 0.71 :1 , or about 1.3 to about 0.77:1 , or about 1.2:1 to about 0.83:1.

[0075] E64. The method of any of the preceding embodiments, further comprising, before the step of reacting, mixing said activated saccharide and carrier protein in the absence of reducing agent.

[0076] E65. The method of E64, further comprising adding said reducing agent to said mixture.

[0077] E66. The method of E65, wherein the amount of reducing agent which is added to the mixture is at least or about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1 , 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 molar equivalents, or ranges from about 0.01 to about 20 molar equivalents.

[0078] E67. The method of any of the preceding embodiments, further comprising, after the step of reacting, terminating the reaction by addition of a capping agent.

[0079] E68. The method of E67, wherein said capping agent is sodium borohydride.

[0080] E69. The method of E67-E68, wherein the amount of capping agent which is added to the reaction is at least or about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1 , 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 11 , 12, 13, 14, 15,

[0081] 16, 17, 18, 19, or 20 molar equivalents, or ranges from about 0.01 to about 20 molar equivalents.

[0082] E70. The method of any of the preceding embodiments, wherein the concentration of carrier protein in said reaction is at least or about 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 ,

[0083] 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3,

[0084] 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5,

[0085] 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9, 10, 11 , 12, 13, 14, 15, 16,

[0086] 17, 18, 19, or 20 g / L, or ranges from about 1.0 g / L to about 20 g / L.

[0087] E71. The method of any of the preceding embodiments, wherein the concentration of activated saccharide in said reaction is at least or about 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,

[0088] 2.1. 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2,

[0089] 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4,

[0090] 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 9, 10, 11 , 12, 13, 14, 15,

[0091] 16, 17, 18, 19, or 20 g / L, or ranges from about 1.0 g / L to about 20 g / L.

[0092] E72. The method of any of the preceding embodiments, wherein the concentration of activated saccharide in said reaction is at least 1 g / L, the carrier protein is CRM197, and the concentration of CRM197 in said reaction is at least 1 g / L.

[0093] E73. The method of any of the preceding embodiments, further comprising purifying the glycoconjugate.

[0094] E74. The method of E73, wherein glycoconjugate is purified by ultrafiltration and / or diafiltration.

[0095] E75. The method of any of the preceding embodiments, wherein the weight average molecular weight of the glycoconjugate in a preparation of said glycoconjugate ranges from about 250 kDa to about 20000 kDa.

[0096] E76. The method of any of the preceding embodiments, wherein the degree of conjugation of the carrier protein in a preparation of said glycoconjugate ranges from about 2 to about 30.

[0097] E77. The method of any of the preceding embodiments, wherein the ratio of saccharide to carrier protein in a preparation of said glycoconjugate ranges from about 0.5 to about 3.0. E78. The method of any of the preceding embodiments, wherein the percentage of free saccharide in a preparation of said glycoconjugate is not more than 30%.

[0098] E79. The method of any of the preceding embodiments, wherein the number of covalent linkages between carrier protein and saccharide in a preparation of said glycoconjugate ranges from about 1 linkage per 1 repeat unit to about 1 linkage per 100 repeat units.

[0099] E80. The method of any of the preceding embodiments, wherein at least 30% of the the glycoconjugate in a preparation of said glycoconjugate has Kd less than or equal to 0.3 as determined using a CL-4B size exclusion column.

[0100] E81. The method of any of the preceding embodiments, wherein the yield, after purification of said glycoconjugate, is at least 45%.

[0101] E82. A glycoconjugate prepared by the method of any one of embodiments E1-E81.

[0102] E83. An immunogenic composition comprising the glycoconjugate of E82.

[0103] E84. A method of preventing or treating a disease or disorder in a subject, comprising administering to a subject in need of prevention or treatment of a disease or disorder a prophylactically or therapeutically effective amount of the immunogenic composition of E83.

[0104] E85. Use of the glycoconjugate of E82 in the preparation of a medicament for the prevention or treatment of a disease or disorder in a subject.

[0105] DETAILED DESCRIPTION

[0106] The inventors have surprisingly discovered novel and inventive methods and compositions for preparing glycoconjugates of activated bacterial capsular saccharides and carrier protein by reductive amination in aprotic solvent which overcome the solubility challenges encountered using existing methods. Use of the new methods and compositions to prepare glycoconjugates surprisingly permits conjugation of certain saccharides and carrier protein by reductive amination in aprotic solvent which has heretofore been impossible and leads to surprising improvements in the quality and yield of certain glycoconjugates which has heretofore been suboptimal. This advance provides more reliable and efficient methods of preparing glycoconjugates and immunogenic compositions containing such glycoconjugates, including vaccines, which may be used to treat or prevent diseases or disorders caused by a bacterial infection.

[0107] Saccharides

[0108] As used herein, the term “saccharide” refers to polysaccharide or oligosaccharide, and unless otherwise specified, may be used interchangeably. In some embodiments, a saccharide is an oligosaccharide. Oligosaccharides can have a low number of repeat units (typically 5-15 repeat units) and are typically derived synthetically or by hydrolysis of polysaccharides. In other embodiments though, all of 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 and methods of the present invention. Therefore, in some embodiments of the present invention, the saccharide is a polysaccharide.

[0109] In some embodiments, saccharides of the disclosure are chemically identical to saccharides which are produced by certain bacteria to form bacterial capsules, and may be referred to as bacterial capsular saccharide (or polysaccharide) or just capsular saccharide (or polysaccharide). Capsules are found in a variety of pathogenic bacteria of medical importance and are largely composed of polysaccharides. The chemical structure of the repeat units of such polysaccharides typically varies among different species of bacteria, and even within the same species of bacteria, different stains may produce capsules comprising polysaccharides which differ substantially in chemical structure.

[0110] Capsular saccharides can be prepared by a variety of techniques known to those of ordinary skill in the art, such as from bacterial cells grown in culture, or by chemical synthetic routes which do not directly involve living cells.

[0111] In some embodiments, bacterial capsular saccharides for use in preparing glycoconjugates of the disclosure are first prepared from 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 Culture Collection (ATCC, Manassas, VA USA) or the Streptococcal Reference Laboratory (Centers for Disease Control and Prevention, Atlanta, GA USA)) or clinical specimens.

[0112] Bacterial capsular saccharides can be obtained directly from bacteria using isolation procedures known 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 W02008 / 118752). They can also be produced using synthetic protocols known to the man skilled in the art.

[0113] In case the bacterial capsular saccharide is obtained directly from bacteria, the bacterial cells can be grown in a medium. Following fermentation of bacterial cells that produce the capsular saccharide, the bacterial 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 W02008 / 118752). The purified capsular saccharide can then be used for the preparation of immunogenic conjugates.

[0114] 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).

[0115] In some embodiments, the capsular saccharide used in the method of making or part of the glycoconjugate of the present invention is a capsular saccharide from a pathogenic bacteria. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from a pathogenic Streptococcus, a pathogenic Staphylococcus, a pathogenic Enterococcus, a pathogenic Bacillus, a pathogenic Corynebacterium, a pathogenic Listeria, a pathogenic Erysipelothrix, a pathogenic Clostridium, a pathogenic Haemophilus, a pathogenic Neisseria or a pathogenic Escherichia. In other embodiments, the capsular saccharide used in the present invention is a capsular saccharide from a pathogenic Streptococcus, a pathogenic Neisseria or a pathogenic Escherichia.

[0116] 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; Brucella suis; Burkholderia mallei (formally Pseudomonas mallei)', Burkholderia pseudomallei (formerly Pseudomonas pseudomallei)', Campylobacter jejuni; Chlamydia psittaci; Chlamydia trachomatis, Clostridium botulinum; Clostridium dificile; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (Heartwater); Coxiella burnetii; Enterococcus faecalis; Enterovirulent Escherichia coli group (EEC Group) such as Escherichia coli - enterotoxigenic (ETEC), Escherichia coli - enteropathogenic (EPEC), Escherichia coli - O157:H7 enterohemorrhagic (EHEC), and Escherichia coli - enteroinvasive (El EC); 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 ly ticus and other Vibrios; Vibrio vulnificus; Xanthomonas oryzae; Xylella fastidiosa (citrus variegated chlorosis strain); Yersinia enterocolitica and Yersinia pseudotuberculosis', or Yersinia pestis. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Enterococcus faecalis, Escherichia coli, Staphylococcus aureus or Streptococcus.

[0117] In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Haemophilus influenzae, Neisseria meningitidis, S. pneumoniae, S. pyogenes, S. agalactiae, Group C & G Streptococci or Escherichia coli. In other embodiments, the capsular saccharide used 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 present invention is a capsular saccharide from S. pneumoniae or S. agalactiae. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from S. pneumoniae.

[0118] 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.

[0119] 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 the present invention is a capsular saccharide from is Haemophilus influenzae type b.

[0120] In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Neisseria meningitidis. In some embodiments the capsular saccharide used in the present invention is a capsular saccharide from N. meningitidis serogroup A (MenA), N. meningitidis serogroup W135 (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 present invention is a capsular saccharide from N. meningitidis serogroup A (MenA). In some embodiments the capsular saccharide used in the present invention is a capsular saccharide from N. meningitidis serogroup W135 (MenW135). In some embodiments the capsular saccharide used in the present invention is a capsular saccharide from N. meningitidis serogroup Y (MenY). In some embodiments the capsular saccharide 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 from N. meningitidis serogroup X (MenX).

[0121] 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.

[0122] In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus agalactiae (Group B streptococcus (GBS)). In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from GBS type la, lb, II, III, IV, V, VI, VII or VIII. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from GBS types la, lb, II, III or V.

[0123] 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 present invention 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 some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Uropathogenic Escherichia coli (LIPEC).

[0124] In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide 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 present invention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting of serotypes O6:K2:H1 and O18:K1 :H7. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype selected from the group consisting of serotypes O45:K1 , O17:K52:H18, O19:H34 and O7:K1. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O104:H4. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O1 :K12:H7. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O127:H6. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O139:H28. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O128:H2.

[0125] In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Steptococcus pneumoniae. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from a Streptococcus pneumoniae serotype 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, 38, 72 and 73, others being possible.

[0126] In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 1. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 2. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 3. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 4. In some embodiments, the capsular 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 capsular saccharide from Streptococcus pneumoniae serotype 6A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 6B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 7C. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 7F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 8. In some embodiments, the capsular saccharide used in the present invention 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 Streptococcus pneumoniae serotype 9N. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 10A. In some embodiments, the capsular 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 capsular saccharide from Streptococcus pneumoniae serotype 11 A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 12F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 14. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 15A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 15B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 15C. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 16F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 17F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 18C. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 19A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 19F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 20. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 20B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 21. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 22A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 22F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 23A. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 23B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 23F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 24B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 24F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 27. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 29. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 31. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 33B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 33F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 34. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 35B. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 35F. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 38. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 72. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus pneumoniae serotype 73.

[0127] Carrier Protein

[0128] Glycoconjugates of the disclosure comprise a carrier protein to which saccharide is conjugated.

[0129] In some embodiments, the carrier protein of the glycoconjugate is an antigenic nontoxic variant of diphtheria toxin, including those referred to with the appelation “cross-reactive material” (CRM), including the version known as CRM197 and others, such as CRM176, CRM228, and CRM45 (llchida et al. (1973) J. Biol. Chem. 218:3838-3844; Giannini, G, et al., (1984) Nuc. Acids Res. 12(10):4063-4069); CRMg, CRM102, CRM103 or CRM ?; 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, Gin or Ser and / or Ala 158 to Gly 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.

[0130] In some embodiments, the carrier protein of the glycoconjugate is CRM197. The CRM197 protein is a nontoxic form of diphtheria toxin but is reportedly immunologically indistinguishable from the diphtheria toxin. CRM197 can be produced by Corynebacterium diphtheriae infected by the nontoxigenic phage pi97tox-created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (llchida 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 CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. Further details about CRM197 and production thereof can be found, e.g., in U.S. Patent No. 5,614,382. In some other embodiments, the carrier protein is a variant of CRM197 protein in which one or more amino acids of the canonical sequence is altered, such as by addition, deletion, or substitution.

[0131] 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 of CRM197 obtained via expression by genetically recombinant E. coli (see, e.g., CN103495161).

[0132] In some embodiments, the carrier protein of the glycoconjugate can be a carrier protein other than a CRM protein, non-limiting examples of which include diphtheria toxoid (DT); tetanus toxoid (TT); fragment C of TT; pneumococcal pneumolysin (ply) (Kuo et al. (1995) Infect Immun 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 (WO 2000 / 37105 and WO 2000 / 39299), and fusions of Pht proteins, for example PhtDE fusions, PhtBE fusions, Pht A-E (WO 2001 / 98334, WO 2003 / 054007, WO 2009 / 000826); OMPC (meningococcal outer membrane protein), which can be extracted from Neisseria meningitidis serogroup B (EP0372501); factor H binding protein (fHBP) from N. meningitidis, including fHBP from subfamily A (such as variant A05) and fHBP from subfamily B (such as variant B01), any of which can be lipidated; a fusion of GNA2091 with fHBP subfamily B, both from N. meningitidis’, fusion of NHBA with GNA1030, both from N. meningitidis', NadA from N. meningitidis', OMV from N. meningitidis’, PorB from N. meningitidis’, PD (Haemophilus influenzae protein D; see, e.g., EP0594610), or immunologically functional equivalents thereof; synthetic peptides (EP0378881 , EP0427347); heat shock proteins (WO 1993 / 17712, WO 1994 / 03208); pertussis proteins (WO 1998 / 58668, EP0471177); cytokines; lymphokines; growth factors or hormones (WO 1991 / 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 Immun 72:4884-4887); pneumococcal surface protein PspA (WO 2002 / 091998); iron uptake proteins (WO 2001 / 72337); toxin A or B of Clostridium difficile (WO 2000 / 61761); transferrin binding proteins; pneumococcal adhesion protein (PsaA); recombinant Pseudomonas aeruginosa exotoxin A, including non-toxic mutants thereof, such as exotoxin A bearing a substution at glutamic acid 553 (Douglas et al. (1987) J. Bacteriol. 169(11):4967-4971); ovalbumin; keyhole limpet hemocyanin (KLH); bovine serum albumin (BSA); purified protein derivative of tuberculin (PPD); inactivated bacterial toxins, such as cholera toxoid (WO 2004 / 083251); Escherichia coli LT; E. coli ST; or exotoxin A from P. aeruginosa.

[0133] In some embodiments, the carrier protein of the glycoconjugate is Streptococcal C5a Peptidase (SCP). In some embodiments, SCP can be from species of p-hemolytic streptococci, for example, from a group A Streptococcus (GAS), such as Streptococcus pyogenes, or from a group B Streptococcus (GBS), such as Streptococcus agalactiae. In some embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP from a GAS (SCPA), or fragment thereof. Non-limiting examples of SCP from GAS include SEQ ID NOs:1 and 2 disclosed in WO 1997 / 26008, and SEQ ID NOs: 1 , 2 and 23 disclosedin WO 2000 / 34487. In other embodiments, the carrier protein of the glycoconjugate is an enzymatically inactive SCP from a GBS (SCPB), or fragment thereof. Non-limiting examples of SCP from GBS include SEQ ID NO:3 disclosed in WO 1997 / 26008 and SEQ ID NO:3 disclosed in WO 2000 / 34487.

[0134] In some embodiments, the carrier protein of the glycoconjugate is a fragment of an SCP, such as a fragment of an SCPA or an SCPB, any of which may be enzymatically inactive, comprising the protease domain, the protease-associated domain (PA domain), and the 3 fibronectin (Fn) type III domains, but not 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, such as a fragment of an SCPA or an SCPB, comprising the protease domain, the PA domain, and 2 of the 3 Fn type III domains, but not any of the export signal pre-sequence, the pro-sequence, or the cell wall anchor domain.

[0135] Methods of preparing glycoconjugates

[0136] Among the embodiments described herein, the present disclosure provides methods of preparing glycoconjugates comprising saccharides conjugated to carrier protein. In some embodiments, the saccharides are bacterial capsular saccharides including, without limitation, bacterial capsular saccharides obtained from pathogenic strains of Streptococcus pneumoniae. In some embodiments, the carrier protein is an antigenic nontoxic variant of diphtheria toxin including, without limitation, those referred to “cross-reactive material” or “CRM” including, without limitation, CRM197, others being possible.

[0137] Saccharides and carrier protein can be conjugated using any method familiar to those of ordinary skill in the art, a non-limiting example of which is chemical conjugation, a non-limiting example of which is reductive amination, other chemical reactions being possible. In some embodiments, saccharides are sized to a target molecular weight or range before carrying out a conjugation reaction. In some embodiments, before carrying out a conjugation reaction, saccharides, whether or not sized, are activated to increase their chemical reactivity with carrier protein. In some embodiments, saccharides are activated by reaction with an oxidizing agent, optionally followed by quenching the oxidation reaction by addition of a quenching agent. In some embodiments, activated saccharides and / or carrier protein can be lyophilized, separately or together. In some embodiments, the lyophilized saccharide and / or carrier protein can be reconstituted in a solvent suitable for the conjugation reaction, after which the reconstituted saccharide and / or carrier protein can be compounded with the other (if not lyophilized together and thereafter reconstituted as such) as well as a reducing agent, causing the activated saccharide and carrier protein to become chemically conjugated.

[0138] Saccharide sizing

[0139] In some embodiments, to generate glycoconjugates with advantageous filterability or other characteristics, immunogenicity and / or yields, sizing of certain saccharides to a target molecular weight range can be performed prior to the conjugation to a carrier protein. Advantageously, the size of the purified capsular saccharide may be reduced while preserving critical features of the structure of the saccharide. Mechanical or chemical sizing may be employed. In some embodiments, the isolated capsular saccharide is not sized before its use in preparing a glycoconjugate as described herein.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] In some embodiments, isolated capsular saccharide can be sized to any suitable weight average molecular weight prior to activation and conjugation. In some embodiments, the weight average molecular weight of isolated capsular saccharide is at least or about 5 kilodaltons (kDa), 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, or 2000 kDa, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0144] Saccharide activation

[0145] In some embodiments, before conjugation with carrier protein, saccharides can be activated by reaction with an oxidizing agent so as to add and / or increase the number of chemical reactive groups. In some embodiments, such reactive groups are capable of reacting directly with corresponding chemical reactive groups in the carrier protein, or reacting with linker molecules which in turn are capable of reacting directly with the carrier protein, in either case producing glycoconjugates.

[0146] In some embodiments, the oxidizing agent is any chemical, compound, or agent capable of oxidizing a terminal hydroxyl present in a saccharide to an aldehyde. In some embodiments, the oxidizing agent is periodate. As used herein, unless otherwise further specified, the term “periodate” refers to periodate and periodic acid, and includes both metaperiodate (IO4 ) and orthoperiodate (IOe5) and the various salts of periodate (e.g., sodium periodate, potassium periodate, sodium metaperiodate, potassium metaperiodate, sodium orthoperiodate, and potassium orthoperiodate). In some embodiments, the oxidizing agent is periodate in the presence bivalent cations (e.g., Mg2+, Ca2+). Without wishing to be bound by theory, when a saccharide reacts with periodate, periodate can oxidize vicinal hydroxyl groups to form a carbonyl or aldehyde group, resulting in cleavage of a C-C bond. In some embodiments, the saccharide which is activated is a bacterial capsular saccharide from a Streptococcus pneumoniae serotype including but not limited to serotypes 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11 A, 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.

[0147] In some embodiments, hydroxyl groups within the saccharide can be activated by TEMPO-mediated oxidation. In some embodiments, the saccharide which is activated in this way is bacterial capsular saccharide from S. pneumoniae serotype 12F, with others being possible.

[0148] In some embodiments, hydroxyl groups within the saccharide can be activated by eTEC- mediated activation, in which saccharide is reacted with CDT, followed by reaction with cystamine dihydrochloride, which introduces a dimeric linker structrure that can thereafter be reduced for conjugation with activated carrier protein. In some embodiments, the saccharide which is activated in this way is bacterial capsular saccharide from S. pneumoniae serotype 33F, with others being possible.

[0149] In some embodiments, saccharide is reacted with an oxidizing agent, such as periodate, under conditions chosen to result in a desired degree of saccharide activation. In some embodiments, saccharide is activated by reacting the saccharide with any suitable amount of the oxidizing agent, such as periodate, which, in some embodiments, is at least or about 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1 , 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 molar equivalents of oxidizing agent to saccharide or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0150] In some embodiments, saccharide is activated by reacting the saccharide with oxidizing agent, such as periodate, for any suitable amount of time, which, in some embodiments, is at least or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 hours, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0151] In some embodiments, saccharide is activated by reacting the saccharide with oxidizing agent, such as periodate, at any suitable temperature, which, in some embodiments, is at least or about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C,40°C, 41 °C, 42°C, 43°C, 44°C, or 45°C, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0152] In some embodiments, saccharide is activated by reacting the saccharide with oxidizing agent, such as periodate, in a reaction mixture comprising any buffer, non-limiting examples of which include sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), or Bis-Tris, others being possible. In some embodiments, saccharide is activated by reacting the saccharide with oxidizing agent, such as periodate, in a reaction mixture comprising a buffer at any suitable pH, which, in some embodiments, is at least or about a pH of 2.0, 2.5, 3.0, 3.5, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.9, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 9.0, 9.5, or 10.0, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0153] In some embodiments, once saccharide has been reacted with oxidizing agent, such as periodate, to a desired degree, the reaction with the oxidizing agent can be quenched by addition of a quenching agent to the reaction. In some embodiments, the quenching agent is a vicinal diol, a 1 ,2-aminoalcohol, glycerol, ethylene glycol, propan-1 , 2-diol, butan-1 ,2-diol, butan-2,3-diol, ascorbic acid, glutathione, a sulfite, a bisulfate, a dithionite, a metabisulfite, a thiosulfate, a phosphite, a hypophosphite, or phosphorous acid. In some embodiments, the quenching agent is selected from sodium and potassium salts of sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphite, or hypophosphite. In some embodiments embodiment, the quenching agent is a 1 ,2-aminoalcohol of formula (I): where R1is selected from H, methyl, ethyl, propyl or isopropyl. In some embodiments, the quenching agent is a compound comprising two vicinal hydroxyl groups (vicinal diols), i.e., two hydroxyl groups covalently linked to two adjacent carbon atoms. In some embodiments, such compounds have the formula (II): where R1and R2are each independently selected from H, methyl, ethyl, propyl or isopropyl. In some embodiments, the quenching agent is an amino acid including, for example, serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.

[0154] After activating saccharide, the degee of activation (also called degree of oxidation) of activated saccharide can be determined according to methods familiar to those of ordinary skill in the art. In some embodiments, the degree of activation of an activated saccharide is at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.

[0155] After activating saccharide, the weight average molecular weight (Mw) of activated saccharide can be determined according to methods familiar to those of ordinary skill in the art. In some embodiments, the weight average molecular weight (Mw) of activated saccharide before conjugation is at least or about 5 kilodaltons (kDa), 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa, 1200 kDa, 1250 kDa, 1300 kDa, 1350 kDa, 1400 kDa, 1450 kDa, 1500 kDa, 1550 kDa, 1600 kDa, 1650 kDa, 1700 kDa, 1750 kDa, 1800 kDa, 1850 kDa, 1900 kDa, 1950 kDa, 2000 kDa, 2500 kDa, 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range. For example, in some non-limiting embodiments, the weight average molecular weight (Mw) of activated saccharide can range from about 10-5000 kDa, 10-4000 kDa, 10-3000 kDa, 10-2000 kDa, 10-1000 kDa, 50-5000 kDa, 50- 4000 kDa, 50-3000 kDa, 50-2000 kDa, 50-1000 kDa, 50-900 kDa, 50-800 kDa, 50-700 kDa, 50- 600 kDa, 50-500 kDa, 50-400 kDa, 50-300 kDa, 50-200 kDa, 50-100 kDa, 75-5000 kDa, 75-4000 kDa, 75-3000 kDa, 75-2000 kDa, 75-1000 kDa, 75-900 kDa, 75-800 kDa, 75-700 kDa, 75-600 kDa, 75-500 kDa, 75-400 kDa, 75-300 kDa, 75-200 kDa, 75-100 kDa, 100-5000 kDa, 100-4000 kDa, 100-3000 kDa, 100-2000 kDa, 100-1000 kDa, 100-900 kDa, 100-800 kDa, 100-700 kDa, 100-600 kDa, 100-500 kDa, 100-400 kDa, 100-300 kDa, 100-200 kDa, 150-5000 kDa, 150-4000 kDa, 150-3000 kDa, 150-2000 kDa, 150-1000 kDa, 150-900 kDa, 150-800 kDa, 150-700 kDa, 150-600 kDa, 150-500 kDa, 150-400 kDa, 150-300 kDa, 150-200 kDa, 200-5000 kDa, 200-4000 kDa, 200-3000 kDa, 200-2000 kDa, 200-1000 kDa, 200-900 kDa, 200-800 kDa, 200-700 kDa, 200-600 kDa, 200-500 kDa, 200-400 kDa, 200-300 kDa, 250-5000 kDa, 250-4000 kDa, 250-3000 kDa, 250-2000 kDa, 250-1000 kDa, 250-900 kDa, 250-800 kDa, 250-700 kDa, 250-600 kDa, 250- 500 kDa, 250-400 kDa, 250-300 kDa, 300-5000 kDa, 300-4000 kDa, 300-3000 kDa, 300-2000 kDa, 300-1000 kDa, 300-900 kDa, 300-800 kDa, 300-700 kDa, 300-600 kDa, 300-500 kDa, 300- 400 kDa, 350-5000 kDa, 350-4000 kDa, 350-3000 kDa, 350-2000 kDa, 350-1000 kDa, 350-900 kDa, 350-800 kDa, 350-700 kDa, 350-600 kDa, 350-500 kDa, 350-400 kDa, 400-5000 kDa, 400- 4000 kDa, 400-3000 kDa, 400-2000 kDa, 400-1000 kDa, 400-900 kDa, 400-800 kDa, 400-700 kDa, 400-600 kDa, 400-500 kDa, 450-5000 kDa, 450-4000 kDa, 450-3000 kDa, 450-2000 kDa, 450-1000 kDa, 450-900 kDa, 450-800 kDa, 450-700 kDa, 450-600 kDa, 450-500 kDa, 500-5000 kDa, 500-4000 kDa, 500-3000 kDa, 500-2000 kDa, 500-1000 kDa, 500-900 kDa, 500-800 kDa, 500-700 kDa, 500-600 kDa, 550-5000 kDa, 550-4000 kDa, 550-3000 kDa, 550-2000 kDa, 550- 1000 kDa, 550-900 kDa, 550-800 kDa, 550-700 kDa, 550-600 kDa, 600-5000 kDa, 600-4000 kDa, 600-3000 kDa, 600-2000 kDa, 600-1000 kDa, 600-900 kDa, 600-800 kDa, 600-700 kDa, 650-5000 kDa, 650-4000 kDa, 650-3000 kDa, 650-2000 kDa, 650-1000 kDa, 650-900 kDa, OSO- SOO kDa, 650-700 kDa, 700-5000 kDa, 700-4000 kDa, 700-3000 kDa, 700-2000 kDa, 700-1000 kDa, 700-900 kDa, 700-800 kDa, 750-5000 kDa, 750-4000 kDa, 750-3000 kDa, 750-2000 kDa, 750-1000 kDa, 750-900 kDa, 750-800 kDa, 800-5000 kDa, 800-4000 kDa, 800-3000 kDa, 800- 2000 kDa, 800-1000 kDa, 800-900 kDa, 850-5000 kDa, 850-4000 kDa, 850-3000 kDa, 850-2000 kDa, 850-1000 kDa, 850-900 kDa, 900-5000 kDa, 900-4000 kDa, 900-3000 kDa, 900-2000 kDa, 900-1000 kDa, 950-5000 kDa, 950-4000 kDa, 950-3000 kDa, 950-2000 kDa, 950-1000 kDa, 1000-5000 kDa, 1000-4000 kDa, 1000-3000 kDa, 1000-2000 kDa, 1200-5000 kDa, 1200-4000 kDa, 1200-3000 kDa, 1200-2000 kDa, 1300-5000 kDa, 1300-4000 kDa, 1300-3000 kDa, 1300- 2000 kDa, 1400-5000 kDa, 1400-4000 kDa, 1400-3000 kDa, 1400-2000 kDa, 1500-5000 kDa, 1500-4000 kDa, 1500-3000 kDa, 1500-2000 kDa, 1600-5000 kDa, 1600-4000 kDa, 1600-3000 kDa, 1600-2000 kDa, 1700-5000 kDa, 1700-4000 kDa, 1700-3000 kDa, 1700-2000 kDa, 1800- 5000 kDa, 1800-4000 kDa, 1800-3000 kDa, 1800-2000 kDa, 1900-5000 kDa, 1900-4000 kDa, 1900-3000 kDa, 1900-2000 kDa, 2000-5000 kDa, 2000-4000 kDa, 2000-3000 kDa, 2100-5000 kDa, 2100-4000 kDa, 2100-3000 kDa, 2200-5000 kDa, 2200-4000 kDa, 2200-3000 kDa, 2300- 5000 kDa, 2300-4000 kDa, 2300-3000 kDa, 2400-5000 kDa, 2400-4000 kDa, 2400-3000 kDa, 2500-5000 kDa, 2500-4000 kDa, 2500-3000 kDa, 2600-5000 kDa, 2600-4000 kDa, 2600-3000 kDa, 2700-5000 kDa, 2700-4000 kDa, 2700-3000 kDa, 2800-5000 kDa, 2800-4000 kDa, 2800- 3000 kDa, 2900-5000 kDa, 2900-4000 kDa, 2900-3000 kDa, 3000-5000 kDa, 3000-4000 kDa, 3100-5000 kDa, 3100-4000 kDa, 3200-5000 kDa, 3200-4000 kDa, 3300-5000 kDa, 3300-4000 kDa, 3400-5000 kDa, 3400-4000 kDa, 3500-5000 kDa, 3500-4000 kDa, 3600-5000 kDa, 3600- 4000 kDa, 3700-5000 kDa, 3700-4000 kDa, 3800-5000 kDa, 3800-4000 kDa, 3900-5000 kDa, 3900-4000 kDa, 4000-5000 kDa, 4100-5000 kDa, 4200-5000 kDa, 4300-5000 kDa, 4400-5000 kDa, 4500-5000 kDa, 4600-5000 kDa, 4700-5000 kDa, 4800-5000 kDa, 4900-5000 kDa, or some other range.

[0156] In some embodiments, depending on the chemical reactive groups which had been introduced into the saccharide by activation, carrier protein can also be activated chemically before carrying out conjugation.

[0157] Lyophilization

[0158] In some embodiments, before conjugation, activated saccharide and / or carrier protein are lyophilized, either separately (discrete lyophilization) or together (co-lyophilization). Lyophilization can be carried out using any method known to those of ordinary skill in the art, a non-limiting example of which is shell lyophilization. In some embodiments, after lyophilization and before conjugation, for example, by reductive amination, either or both lyophilized activated saccharide and / or carrier protein, as the case may be, can be reconstituted in a solvent suitable for carrying out the conjugation reaction. In the case where each of the activated saccharide and carrier protein had been lyophilized separately, both lyophilizates can be reconstituted in solvent and then compounded together before conjugation. In the case where both the activated saccharide and carrier protein had been lyophilized together, then the combined lyophilizate can be reconstituted in solvent before proceeding to carry out the conjugation reaction. In other embodiments where one of activated saccharide or carrier protein had been lyophilized and the other ingredient maintained in solution (whether liquid or frozen) and not lyophilized, then the component which had been lyophilized can be reconstituted in solvent and then compounded with the component maintained in solution or, where the component maintained in solution had been maintained in solvent suitable for carrying out the conjugation reaction, then such solution can be used to reconstitute the lyophilized component.

[0159] In some embodiments, activated saccharide and / or carrier protein (separately or together) can be maintained in a solution (lyophilization solution) which is subjected to lyophilization, producing a lyophilizate comprising either or both components. In some embodiments, the lyophilization solution can comprise one or more ingredients, such as salts, buffers, sugars, and other chemicals, compounds, or agents, affecting the properties of the lyophilizate, such as stability or solubility. For example, it was surprisingly discovered that lyophilization of certain carrier proteins, such as CRM197, in buffer containing sodium ions produced a lyophilizate which is poorly soluble in aprotic solvents, such as dimethylsulfoxide (DMSO), e.g., below about 3 g / L, which can be disadvantageous for carrying out certain conjugation reactions, such as reductive amination in an aprotic versus aqueous solvent. Even more surprisingly, given the similar physical and chemical nature of the cations, the inventors discovered that lyophilization of carrier protein, such as CRM197, in the presence of potassium ions dramatically increased carrier solubility in aprotic solvent making it feasible to conjugate carrier protein and activated saccharide in aprotic solvents at higher concentrations than was otherwise understood to be possible. Based on this surprising discovery, the disclosure provides methods and compositions for lyophilizing carrier protein, alone or with activated saccharide, in the presence of K+ions, thereby enhancing carrier protein solubility upon reconstitution with aprotic solvent prior to carrying out the conjugation reaction. Such methods and compositions advantageously improve the efficiency by which glycoconjugates can be produced, as well as their physical, chemical and biological properties. In some embodiments, the carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein, including without limitation CRM197, the saccharide is a bacterial capsular saccharide, including without limitation a bacterial capsular saccharide from a pathogenic strain of Streptococcus pneumoniae, the aprotic solvent is DMSO, and the conjugation reaction is reductive amination.

[0160] In some embodiments, lyophilization solution comprises one or more compounds which function as a pH buffer. In some embodiments, the buffer is a sodium phosphate buffer or a potassium phosphate buffer. Sodium or potassium phosphate buffers can be prepared according to the knowledge of those ordinarily skilled in the art. For example, as known in the art, sodium phosphate buffer can be prepared from stock solutions of sodium phosphate monobasic and sodium phosphate dibasic in proportions that determine the ultimate pH of the solution. Similarly, potassium phosphate buffer can be prepared from stock solutions of potassium phosphate monobasic and potassium phosphate dibasic in proportions that determine the ultimate pH of the solution. In some embodiments, lyophilization solution comprises predominantly or exclusively potassium phosphate as the buffer, thereby providing a source of potassium ions while simultaneously reducing or eliminating the buffer as a source of sodium ions. In some other embodiments, lyophilization solution can comprise different organic or inorganic salts, such as KCI, which provide potassium ions upon disassociation as an additional or alternative source of such ions than the components of potassium phosphate buffer. Inclusion of such other potassium salts allows the amount of potassium in the lyophilization solution to be increased while maintaining the potassium phosphate buffer concentration steady, if so desired, or to substitute a buffer other than potassium phosphate buffer while providing sufficient potassium to support increased carrier protein solubility in aprotic solvent as described herein. In some embodiments, lyophilization solution comprises potassium as well as sodium ions, in varying proportions, whereas in other embodiments, lyophilization solution comprises potassium as the predominant monovalent cation and a minimum of sodium ions, whatever the source, or even just trace amounts of sodium ions as might be expected from the normal level of contaminating sodium ions present in GLP or GMP grade reagents.

[0161] In some embodiments, lyophilization solution further comprises a non-reducing sugar, non limiting examples of which include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinose, isomalt, and verbascose, others being possible. In some embodiments, the lyophilization solution comprises sucrose.

[0162] Lyophilization solution

[0163] Various non-limiting embodiments of lyophilization solution are described further below. pH

[0164] In some embodiments, the pH of the lyophilization solution can be any pH suitable for lyophilizing carrier protein and / or saccharide for subsequent reconstitution to carry out reductive amination. In some embodiments, the pH of the lyophilization solution is at least or about pH 5,

[0165] 5.5, 6, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4,

[0166] 8.5, 9, 9.5, or 10, or value between or range encompassing any of the foregoing specifically enumerated pH values as end points.

[0167] Potassium ion concentration

[0168] In some embodiments, before lyophilization, the lyophilization solution can comprise one or more potassium salts, such that the concentration of potassium ions (K+) in the solution is at least or about 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 millimolar (mM), or concentration between or range encompassing any of the foregoing specifically enumerated concentration values as end points, such as 0.5-90 mM, 1-85 mM, 2-80 mM, 3-75 mM, 4-90 mM,

[0169] 4-85 mM, 4-80 mM, 4-75 mM, 4-70 mM, 4-65 mM, 4-60 mM, 4-55 mM, 4-50 mM, 4-45 mM, 4-40 mM, 4-35 mM, 4-30 mM, 4-25 mM, 4-20 mM, 4-18 mM, 4-16 mM, 4-14 mM, 4-12 mM, 4-10 mM,

[0170] 5-90 mM, 5-85 mM, 5-80 mM, 5-75 mM, 5-70 mM, 5-65 mM, 5-60 mM, 5-55 mM, 5-50 mM, 5-45 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-18 mM, 5-16 mM, 5-14 mM, 5-12 mM,

[0171] 5-10 mM, 6-90 mM, 6-85 mM, 6-80 mM, 6-75 mM, 6-70 mM, 6-65 mM, 6-60 mM, 6-55 mM, 6-50 mM, 6-45 mM, 6-40 mM, 6-35 mM, 6-30 mM, 6-25 mM, 6-20 mM, 6-18 mM, 6-16 mM, 6-14 mM,

[0172] 6-12 mM, 6-10 mM, 7-90 mM, 7-85 mM, 7-80 mM, 7-75 mM, 7-70 mM, 7-65 mM, 7-60 mM, 7-55 mM, 7-50 mM, 7-45 mM, 7-40 mM, 7-35 mM, 7-30 mM, 7-25 mM, 7-20 mM, 7-18 mM, 7-16 mM,

[0173] 7-14 mM, 7-12 mM, 7-10 mM, 8-90 mM, 8-85 mM, 8-80 mM, 8-75 mM, 8-70 mM, 8-65 mM, 8-60 mM, 8-55 mM, 8-50 mM, 8-45 mM, 8-40 mM, 8-35 mM, 8-30 mM, 8-25 mM, 8-20 mM, 8-18 mM,

[0174] 8-16 mM, 8-14 mM, 8-12 mM, 8-10 mM, 8.5-9.5 mM, 7.5-10.5 mM, 7-11 mM, 6.5-11.5 mM, 5.5- 12.5 mM, 5-13 mM, or 4.5-13.5 mM, or some other range or value. In any of the foregoing embodiments, the potassium salt can be potassium phosphate monobasic (including hydrates thereof), and / or potassium phosphate dibasic (including hydrates thereof), and / or potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof), and / or potassium phosphate dibasic (including hydrates thereof), but no added potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof) and potassium phosphate dibasic (including hydrates thereof), and / or potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof) and potassium phosphate dibasic (including hydrates thereof), but no added potassium chloride.

[0175] Potassium and sodium ion concentration

[0176] In some embodiments, before lyophilization, the lyophilization solution comprises one or more potassium salts, such that the concentration of potassium ions (K+) in the solution is at least or about 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 millimolar (mM), or value between or range encompassing any of the foregoing specifically enumerated concentration values as end points, such as 0.5-90 mM, 1-85 mM, 2-80 mM, 3-75 mM, 4-90 mM,

[0177] 4-85 mM, 4-80 mM, 4-75 mM, 4-70 mM, 4-65 mM, 4-60 mM, 4-55 mM, 4-50 mM, 4-45 mM, 4-40 mM, 4-35 mM, 4-30 mM, 4-25 mM, 4-20 mM, 4-18 mM, 4-16 mM, 4-14 mM, 4-12 mM, 4-10 mM,

[0178] 5-90 mM, 5-85 mM, 5-80 mM, 5-75 mM, 5-70 mM, 5-65 mM, 5-60 mM, 5-55 mM, 5-50 mM, 5-45 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-18 mM, 5-16 mM, 5-14 mM, 5-12 mM,

[0179] 5-10 mM, 6-90 mM, 6-85 mM, 6-80 mM, 6-75 mM, 6-70 mM, 6-65 mM, 6-60 mM, 6-55 mM, 6-50 mM, 6-45 mM, 6-40 mM, 6-35 mM, 6-30 mM, 6-25 mM, 6-20 mM, 6-18 mM, 6-16 mM, 6-14 mM,

[0180] 6-12 mM, 6-10 mM, 7-90 mM, 7-85 mM, 7-80 mM, 7-75 mM, 7-70 mM, 7-65 mM, 7-60 mM, 7-55 mM, 7-50 mM, 7-45 mM, 7-40 mM, 7-35 mM, 7-30 mM, 7-25 mM, 7-20 mM, 7-18 mM, 7-16 mM,

[0181] 7-14 mM, 7-12 mM, 7-10 mM, 8-90 mM, 8-85 mM, 8-80 mM, 8-75 mM, 8-70 mM, 8-65 mM, 8-60 mM, 8-55 mM, 8-50 mM, 8-45 mM, 8-40 mM, 8-35 mM, 8-30 mM, 8-25 mM, 8-20 mM, 8-18 mM,

[0182] 8-16 mM, 8-14 mM, 8-12 mM, 8-10 mM, 8.5-9.5 mM, 7.5-10.5 mM, 7-11 mM, 6.5-11.5 mM, 5.5- 12.5 mM, 5-13 mM, or 4.5-13.5 mM, or some other range or value. In any of these embodiments, the lyophilization solution can, at the same time, comprise less than 25 mM of sodium ions (Na+), whatever the source, for example, less than 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.25, 0.1 , 0.05 mM sodium ions, or lower amounts of sodium ions, such as not more than trace amounts of sodium ions. In any of the foregoing embodiments, the potassium salt can be potassium phosphate monobasic (including hydrates thereof), and / or potassium phosphate dibasic (including hydrates thereof), and / or potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof), and / or potassium phosphate dibasic (including hydrates thereof), but no added potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof) and potassium phosphate dibasic (including hydrates thereof), and / or potassium chloride. In some embodiments, the potassium salt is potassium phosphate monobasic (including hydrates thereof) and potassium phosphate dibasic (including hydrates thereof), but no added potassium chloride.

[0183] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 2 mM K+and less than 15 mM Na+, or at least or about 2 mM K+and less than 14 mM Na+, or at least or about 2 mM K+and less than 13 mM Na+, or at least or about 2 mM K+and less than 12 mM Na+, or at least or about 2 mM K+and less than 11 mM Na+, or at least or about 2 mM K+and less than 10 mM Na+, or at least or about 2 mM K+and less than 9 mM Na+, or at least or about 2 mM K+and less than 8 mM Na+, or at least or about 2 mM K+and less than 7 mM Na+, or at least or about 2 mM K+and less than 6 mM Na+, or at least or about 2 mM K+and less than 5 mM Na+, or at least or about 2 mM K+and less than 4 mM Na+, or at least or about 2 mM K+and less than 3 mM Na+, or at least or about 2 mM K+and less than 2 mM Na+, or at least or about 2 mM K+and less than 1 mM Na+, or at least or about 2 mM K+and less than 0.5 mM Na+.

[0184] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 3 mM K+and less than 15 mM Na+, or at least or about 3 mM K+and less than 14 mM Na+, or at least or about 3 mM K+and less than 13 mM Na+, or at least or about 3 mM K+and less than 12 mM Na+, or at least or about 3 mM K+and less than 11 mM Na+, or at least or about

[0185] 3 mM K+and less than 10 mM Na+, or at least or about 3 mM K+and less than 9 mM Na+, or at least or about 3 mM K+and less than 8 mM Na+, or at least or about 3 mM K+and less than 7 mM Na+, or at least or about 3 mM K+and less than 6 mM Na+, or at least or about 3 mM K+and less than 5 mM Na+, or at least or about 3 mM K+and less than 4 mM Na+, or at least or about 3 mM K+and less than 3 mM Na+, or at least or about 3 mM K+and less than 2 mM Na+, or at least or about 3 mM K+and less than 1 mM Na+, or at least or about 3 mM K+and less than 0.5 mM Na+.

[0186] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 4 mM K+and less than 15 mM Na+, or at least or about 4 mM K+and less than 14 mM Na+, or at least or about 4 mM K+and less than 13 mM Na+, or at least or about 4 mM K+and less than 12 mM Na+, or at least or about 4 mM K+and less than 11 mM Na+, or at least or about

[0187] 4 mM K+and less than 10 mM Na+, or at least or about 4 mM K+and less than 9 mM Na+, or at least or about 4 mM K+and less than 8 mM Na+, or at least or about 4 mM K+and less than 7 mM Na+, or at least or about 4 mM K+and less than 6 mM Na+, or at least or about 4 mM K+and less than 5 mM Na+, or at least or about 4 mM K+and less than 4 mM Na+, or at least or about 4 mM K+and less than 3 mM Na+, or at least or about 4 mM K+and less than 2 mM Na+, or at least or about 4 mM K+and less than 1 mM Na+, or at least or about 4 mM K+and less than 0.5 mM Na+.

[0188] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 5 mM K+and less than 15 mM Na+, or at least or about 5 mM K+and less than 14 mM Na+, or at least or about 5 mM K+and less than 13 mM Na+, or at least or about 5 mM K+and less than 12 mM Na+, or at least or about 5 mM K+and less than 11 mM Na+, or at least or about

[0189] 5 mM K+and less than 10 mM Na+, or at least or about 5 mM K+and less than 9 mM Na+, or at least or about 5 mM K+and less than 8 mM Na+, or at least or about 5 mM K+and less than 7 mM Na+, or at least or about 5 mM K+and less than 6 mM Na+, or at least or about 5 mM K+and less than 5 mM Na+, or at least or about 5 mM K+and less than 4 mM Na+, or at least or about 5 mM K+and less than 3 mM Na+, or at least or about 5 mM K+and less than 2 mM Na+, or at least or about 5 mM K+and less than 1 mM Na+, or at least or about 5 mM K+and less than 0.5 mM Na+.

[0190] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 6 mM K+and less than 15 mM Na+, or at least or about 6 mM K+and less than 14 mM Na+, or at least or about 6 mM K+and less than 13 mM Na+, or at least or about 6 mM K+and less than 12 mM Na+, or at least or about 6 mM K+and less than 11 mM Na+, or at least or about

[0191] 6 mM K+and less than 10 mM Na+, or at least or about 6 mM K+and less than 9 mM Na+, or at least or about 6 mM K+and less than 8 mM Na+, or at least or about 6 mM K+and less than 7 mM Na+, or at least or about 6 mM K+and less than 6 mM Na+, or at least or about 6 mM K+and less than 5 mM Na+, or at least or about 6 mM K+and less than 4 mM Na+, or at least or about 6 mM K+and less than 3 mM Na+, or at least or about 6 mM K+and less than 2 mM Na+, or at least or about 6 mM K+and less than 1 mM Na+, or at least or about 6 mM K+and less than 0.5 mM Na+.

[0192] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 7 mM K+and less than 15 mM Na+, or at least or about 7 mM K+and less than 14 mM Na+, or at least or about 7 mM K+and less than 13 mM Na+, or at least or about 7 mM K+and less than 12 mM Na+, or at least or about 7 mM K+and less than 11 mM Na+, or at least or about

[0193] 7 mM K+and less than 10 mM Na+, or at least or about 7 mM K+and less than 9 mM Na+, or at least or about 7 mM K+and less than 8 mM Na+, or at least or about 7 mM K+and less than 7 mM Na+, or at least or about 7 mM K+and less than 6 mM Na+, or at least or about 7 mM K+and less than 5 mM Na+, or at least or about 7 mM K+and less than 4 mM Na+, or at least or about 7 mM K+and less than 3 mM Na+, or at least or about 7 mM K+and less than 2 mM Na+, or at least or about 7 mM K+and less than 1 mM Na+, or at least or about 7 mM K+and less than 0.5 mM Na+.

[0194] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 8 mM K+and less than 15 mM Na+, or at least or about 8 mM K+and less than 14 mM Na+, or at least or about 8 mM K+and less than 13 mM Na+, or at least or about 8 mM K+and less than 12 mM Na+, or at least or about 8 mM K+and less than 11 mM Na+, or at least or about

[0195] 8 mM K+and less than 10 mM Na+, or at least or about 8 mM K+and less than 9 mM Na+, or at least or about 8 mM K+and less than 8 mM Na+, or at least or about 8 mM K+and less than 7 mM Na+, or at least or about 8 mM K+and less than 6 mM Na+, or at least or about 8 mM K+and less than 5 mM Na+, or at least or about 8 mM K+and less than 4 mM Na+, or at least or about 8 mM K+and less than 3 mM Na+, or at least or about 8 mM K+and less than 2 mM Na+, or at least or about 8 mM K+and less than 1 mM Na+, or at least or about 8 mM K+and less than 0.5 mM Na+.

[0196] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 9 mM K+and less than 15 mM Na+, or at least or about 9 mM K+and less than 14 mM Na+, or at least or about 9 mM K+and less than 13 mM Na+, or at least or about 9 mM K+and less than 12 mM Na+, or at least or about 9 mM K+and less than 11 mM Na+, or at least or about

[0197] 9 mM K+and less than 10 mM Na+, or at least or about 9 mM K+and less than 9 mM Na+, or at least or about 9 mM K+and less than 8 mM Na+, or at least or about 9 mM K+and less than 7 mM Na+, or at least or about 9 mM K+and less than 6 mM Na+, or at least or about 9 mM K+and less than 5 mM Na+, or at least or about 9 mM K+and less than 4 mM Na+, or at least or about 9 mM K+and less than 3 mM Na+, or at least or about 9 mM K+and less than 2 mM Na+, or at least or about 9 mM K+and less than 1 mM Na+, or at least or about 9 mM K+and less than 0.5 mM Na+.

[0198] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 10 mM K+and less than 15 mM Na+, or at least or about 10 mM K+and less than 14 mM Na+, or at least or about 10 mM K+and less than 13 mM Na+, or at least or about 10 mM K+and less than 12 mM Na+, or at least or about 10 mM K+and less than 11 mM Na+, or at least or about

[0199] 10 mM K+and less than 10 mM Na+, or at least or about 10 mM K+and less than 9 mM Na+, or at least or about 10 mM K+and less than 8 mM Na+, or at least or about 10 mM K+and less than 7 mM Na+, or at least or about 10 mM K+and less than 6 mM Na+, or at least or about 10 mM K+and less than 5 mM Na+, or at least or about 10 mM K+and less than 4 mM Na+, or at least or about 10 mM K+and less than 3 mM Na+, or at least or about 10 mM K+and less than 2 mM Na+, or at least or about 10 mM K+and less than 1 mM Na+, or at least or about 10 mM K+and less than 0.5 mM Na+.

[0200] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 11 mM K+and less than 15 mM Na+, or at least or about 11 mM K+and less than 14 mM Na+, or at least or about 11 mM K+and less than 13 mM Na+, or at least or about 11 mM K+and less than 12 mM Na+, or at least or about 11 mM K+and less than 11 mM Na+, or at least or about

[0201] 11 mM K+and less than 10 mM Na+, or at least or about 11 mM K+and less than 9 mM Na+, or at least or about 11 mM K+and less than 8 mM Na+, or at least or about 11 mM K+and less than 7 mM Na+, or at least or about 11 mM K+and less than 6 mM Na+, or at least or about 11 mM K+and less than 5 mM Na+, or at least or about 11 mM K+and less than 4 mM Na+, or at least or about 11 mM K+and less than 3 mM Na+, or at least or about 11 mM K+and less than 2 mM Na+, or at least or about 11 mM K+and less than 1 mM Na+, or at least or about 11 mM K+and less than 0.5 mM Na+.

[0202] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 12 mM K+and less than 15 mM Na+, or at least or about 12 mM K+and less than 14 mM Na+, or at least or about 12 mM K+and less than 13 mM Na+, or at least or about 12 mM K+and less than 12 mM Na+, or at least or about 12 mM K+and less than 11 mM Na+, or at least or about

[0203] 12 mM K+and less than 10 mM Na+, or at least or about 12 mM K+and less than 9 mM Na+, or at least or about 12 mM K+and less than 8 mM Na+, or at least or about 12 mM K+and less than 7 mM Na+, or at least or about 12 mM K+and less than 6 mM Na+, or at least or about 12 mM K+and less than 5 mM Na+, or at least or about 12 mM K+and less than 4 mM Na+, or at least or about 12 mM K+and less than 3 mM Na+, or at least or about 12 mM K+and less than 2 mM Na+, or at least or about 12 mM K+and less than 1 mM Na+, or at least or about 12 mM K+and less than 0.5 mM Na+.

[0204] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 13 mM K+and less than 15 mM Na+, or at least or about 13 mM K+and less than 14 mM Na+, or at least or about 13 mM K+and less than 13 mM Na+, or at least or about 13 mM K+and less than 12 mM Na+, or at least or about 13 mM K+and less than 11 mM Na+, or at least or about

[0205] 13 mM K+and less than 10 mM Na+, or at least or about 13 mM K+and less than 9 mM Na+, or at least or about 13 mM K+and less than 8 mM Na+, or at least or about 13 mM K+and less than 7 mM Na+, or at least or about 13 mM K+and less than 6 mM Na+, or at least or about 13 mM K+and less than 5 mM Na+, or at least or about 13 mM K+and less than 4 mM Na+, or at least or about 13 mM K+and less than 3 mM Na+, or at least or about 13 mM K+and less than 2 mM Na+, or at least or about 13 mM K+and less than 1 mM Na+, or at least or about 13 mM K+and less than 0.5 mM Na+.

[0206] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 14 mM K+and less than 15 mM Na+, or at least or about 14 mM K+and less than 14 mM Na+, or at least or about 14 mM K+and less than 13 mM Na+, or at least or about 14 mM K+and less than 12 mM Na+, or at least or about 14 mM K+and less than 11 mM Na+, or at least or about

[0207] 14 mM K+and less than 10 mM Na+, or at least or about 14 mM K+and less than 9 mM Na+, or at least or about 14 mM K+and less than 8 mM Na+, or at least or about 14 mM K+and less than 7 mM Na+, or at least or about 14 mM K+and less than 6 mM Na+, or at least or about 14 mM K+and less than 5 mM Na+, or at least or about 14 mM K+and less than 4 mM Na+, or at least or about 14 mM K+and less than 3 mM Na+, or at least or about 14 mM K+and less than 2 mM Na+, or at least or about 14 mM K+and less than 1 mM Na+, or at least or about 14 mM K+and less than 0.5 mM Na+.

[0208] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 15 mM K+and less than 15 mM Na+, or at least or about 15 mM K+and less than 14 mM Na+, or at least or about 15 mM K+and less than 13 mM Na+, or at least or about 15 mM K+and less than 12 mM Na+, or at least or about 15 mM K+and less than 11 mM Na+, or at least or about

[0209] 15 mM K+and less than 10 mM Na+, or at least or about 15 mM K+and less than 9 mM Na+, or at least or about 15 mM K+and less than 8 mM Na+, or at least or about 15 mM K+and less than 7 mM Na+, or at least or about 15 mM K+and less than 6 mM Na+, or at least or about 15 mM K+and less than 5 mM Na+, or at least or about 15 mM K+and less than 4 mM Na+, or at least or about 15 mM K+and less than 3 mM Na+, or at least or about 15 mM K+and less than 2 mM Na+, or at least or about 15 mM K+and less than 1 mM Na+, or at least or about 15 mM K+and less than 0.5 mM Na+.

[0210] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 16 mM K+and less than 15 mM Na+, or at least or about 16 mM K+and less than 14 mM Na+, or at least or about 16 mM K+and less than 13 mM Na+, or at least or about 16 mM K+and less than 12 mM Na+, or at least or about 16 mM K+and less than 11 mM Na+, or at least or about

[0211] 16 mM K+and less than 10 mM Na+, or at least or about 16 mM K+and less than 9 mM Na+, or at least or about 16 mM K+and less than 8 mM Na+, or at least or about 16 mM K+and less than 7 mM Na+, or at least or about 16 mM K+and less than 6 mM Na+, or at least or about 16 mM K+and less than 5 mM Na+, or at least or about 16 mM K+and less than 4 mM Na+, or at least or about 16 mM K+and less than 3 mM Na+, or at least or about 16 mM K+and less than 2 mM Na+, or at least or about 16 mM K+and less than 1 mM Na+, or at least or about 16 mM K+and less than 0.5 mM Na+.

[0212] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 17 mM K+and less than 15 mM Na+, or at least or about 17 mM K+and less than 14 mM Na+, or at least or about 17 mM K+and less than 13 mM Na+, or at least or about 17 mM K+and less than 12 mM Na+, or at least or about 17 mM K+and less than 11 mM Na+, or at least or about

[0213] 17 mM K+and less than 10 mM Na+, or at least or about 17 mM K+and less than 9 mM Na+, or at least or about 17 mM K+and less than 8 mM Na+, or at least or about 17 mM K+and less than 7 mM Na+, or at least or about 17 mM K+and less than 6 mM Na+, or at least or about 17 mM K+and less than 5 mM Na+, or at least or about 17 mM K+and less than 4 mM Na+, or at least or about 17 mM K+and less than 3 mM Na+, or at least or about 17 mM K+and less than 2 mM Na+, or at least or about 17 mM K+and less than 1 mM Na+, or at least or about 17 mM K+and less than 0.5 mM Na+.

[0214] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 18 mM K+and less than 15 mM Na+, or at least or about 18 mM K+and less than 14 mM Na+, or at least or about 18 mM K+and less than 13 mM Na+, or at least or about 18 mM K+and less than 12 mM Na+, or at least or about 18 mM K+and less than 11 mM Na+, or at least or about

[0215] 18 mM K+and less than 10 mM Na+, or at least or about 18 mM K+and less than 9 mM Na+, or at least or about 18 mM K+and less than 8 mM Na+, or at least or about 18 mM K+and less than 7 mM Na+, or at least or about 18 mM K+and less than 6 mM Na+, or at least or about 18 mM K+and less than 5 mM Na+, or at least or about 18 mM K+and less than 4 mM Na+, or at least or about 18 mM K+and less than 3 mM Na+, or at least or about 18 mM K+and less than 2 mM Na+, or at least or about 18 mM K+and less than 1 mM Na+, or at least or about 18 mM K+and less than 0.5 mM Na+.

[0216] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 19 mM K+ and less than 15 mM Na+, or at least or about 19 mM K+and less than 14 mM Na+, or at least or about 19 mM K+and less than 13 mM Na+, or at least or about 19 mM K+and less than 12 mM Na+, or at least or about 19 mM K+and less than 11 mM Na+, or at least or about 19 mM K+and less than 10 mM Na+, or at least or about 19 mM K+and less than 9 mM Na+, or at least or about 19 mM K+and less than 8 mM Na+, or at least or about 19 mM K+and less than 7 mM Na+, or at least or about 19 mM K+and less than 6 mM Na+, or at least or about 19 mM K+and less than 5 mM Na+, or at least or about 19 mM K+and less than 4 mM Na+, or at least or about 19 mM K+and less than 3 mM Na+, or at least or about 19 mM K+and less than 2 mM Na+, or at least or about 19 mM K+and less than 1 mM Na+, or at least or about 19 mM K+and less than 0.5 mM Na+.

[0217] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 20 mM K+and less than 15 mM Na+, or at least or about 20 mM K+and less than 14 mM Na+, or at least or about 20 mM K+and less than 13 mM Na+, or at least or about 20 mM K+and less than 12 mM Na+, or at least or about 20 mM K+and less than 11 mM Na+, or at least or about

[0218] 20 mM K+and less than 10 mM Na+, or at least or about 20 mM K+and less than 9 mM Na+, or at least or about 20 mM K+and less than 8 mM Na+, or at least or about 20 mM K+and less than 7 mM Na+, or at least or about 20 mM K+and less than 6 mM Na+, or at least or about 20 mM K+and less than 5 mM Na+, or at least or about 20 mM K+and less than 4 mM Na+, or at least or about 20 mM K+and less than 3 mM Na+, or at least or about 20 mM K+and less than 2 mM Na+, or at least or about 20 mM K+and less than 1 mM Na+, or at least or about 20 mM K+and less than 0.5 mM Na+.

[0219] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 25 mM K+and less than 15 mM Na+, or at least or about 25 mM K+and less than 14 mM Na+, or at least or about 25 mM K+and less than 13 mM Na+, or at least or about 25 mM K+and less than 12 mM Na+, or at least or about 25 mM K+and less than 11 mM Na+, or at least or about 25 mM K+and less than 10 mM Na+, or at least or about 25 mM K+and less than 9 mM Na+, or at least or about 25 mM K+and less than 8 mM Na+, or at least or about 25 mM K+and less than 7 mM Na+, or at least or about 25 mM K+and less than 6 mM Na+, or at least or about 25 mM K+and less than 5 mM Na+, or at least or about 25 mM K+and less than 4 mM Na+, or at least or about 25 mM K+and less than 3 mM Na+, or at least or about 25 mM K+and less than 2 mM Na+, or at least or about 25 mM K+and less than 1 mM Na+, or at least or about 25 mM K+and less than 0.5 mM Na+.

[0220] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 30 mM K+and less than 15 mM Na+, or at least or about 30 mM K+and less than 14 mM Na+, or at least or about 30 mM K+and less than 13 mM Na+, or at least or about 30 mM K+and less than 12 mM Na+, or at least or about 30 mM K+and less than 11 mM Na+, or at least or about 30 mM K+and less than 10 mM Na+, or at least or about 30 mM K+and less than 9 mM Na+, or at least or about 30 mM K+and less than 8 mM Na+, or at least or about 30 mM K+and less than 7 mM Na+, or at least or about 30 mM K+and less than 6 mM Na+, or at least or about 30 mM K+and less than 5 mM Na+, or at least or about 30 mM K+and less than 4 mM Na+, or at least or about 30 mM K+and less than 3 mM Na+, or at least or about 30 mM K+and less than 2 mM Na+, or at least or about 30 mM K+and less than 1 mM Na+, or at least or about 30 mM K+and less than 0.5 mM Na+. In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 35 mM K+and less than 15 mM Na+, or at least or about 35 mM K+and less than 14 mM Na+, or at least or about 35 mM K+and less than 13 mM Na+, or at least or about 35 mM K+and less than 12 mM Na+, or at least or about 35 mM K+and less than 11 mM Na+, or at least or about 35 mM K+and less than 10 mM Na+, or at least or about 35 mM K+and less than 9 mM Na+, or at least or about 35 mM K+and less than 8 mM Na+, or at least or about 35 mM K+and less than 7 mM Na+, or at least or about 35 mM K+and less than 6 mM Na+, or at least or about 35 mM K+and less than 5 mM Na+, or at least or about 35 mM K+and less than 4 mM Na+, or at least or about 35 mM K+and less than 3 mM Na+, or at least or about 35 mM K+and less than 2 mM Na+, or at least or about 35 mM K+and less than 1 mM Na+, or at least or about 35 mM K+and less than 0.5 mM Na+.

[0221] In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 40 mM K+and less than 15 mM Na+, or at least or about 40 mM K+and less than 14 mM Na+, or at least or about 40 mM K+and less than 13 mM Na+, or at least or about 40 mM K+and less than 12 mM Na+, or at least or about 40 mM K+and less than 11 mM Na+, or at least or about 40 mM K+and less than 10 mM Na+, or at least or about 40 mM K+and less than 9 mM Na+, or at least or about 40 mM K+and less than 8 mM Na+, or at least or about 40 mM K+and less than 7 mM Na+, or at least or about 40 mM K+and less than 6 mM Na+, or at least or about 40 mM K+and less than 5 mM Na+, or at least or about 40 mM K+and less than 4 mM Na+, or at least or about 40 mM K+and less than 3 mM Na+, or at least or about 40 mM K+and less than 2 mM Na+, or at least or about 40 mM K+and less than 1 mM Na+, or at least or about 40 mM K+and less than 0.5 mM Na+.

[0222] Molar ratio of potassium ions to carrier protein

[0223] In some embodiments, before lyophilization, the molar ratio of the quantity of K+ions (measured in moles) to the quantity of carrier protein (measured in moles) in the lyophilization solution is at least or about 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550: 1 , 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 1:1, 900:1 to 2:1, 800:1 to 3:1, 700:1 to 4:1, 600:1 to 5:1, 500:1 to 10:1, 400:1 to 15:1, 300:1 to 85:1, 300:1 to 80:1, 300:1 to 75:1, 300:1 to 70:1, 300:1 to 65:1, 300:1 to 60:1, 300:1 to 55:1, 300:1 to 50:1, 300: 1 to 45: 1 , 300: 1 to 40: 1 , 300: 1 to 35: 1 , 300: 1 to 30: 1 , 300: 1 to 25: 1 , 300: 1 to 20: 1 , 300: 1 to 15:1, 300:1 to 10:1, 300:1 to 5:1, 300:1 to 4:1, 300:1 to 3:1, 300:1 to 2:1, 300:1 to 1:1, 200:1 to 85:1, 200:1 to 80:1, 200:1 to 75:1, 200:1 to 70:1, 200:1 to 65:1, 200:1 to 60:1, 200:1 to 55:1, 200: 1 to 50: 1 , 200: 1 to 45: 1 , 200: 1 to 40: 1 , 200: 1 to 35: 1 , 200: 1 to 30: 1 , 200: 1 to 25: 1 , 200: 1 to 20:1, 200:1 to 15:1, 200:1 to 10:1, 200:1 to 5:1, 200:1 to 4:1, 200:1 to 3:1, 200:1 to 2:1, 200:1 to 1:1, 150:1 to 85:1, 150:1 to 80:1, 150:1 to 75:1, 150:1 to 70:1, 150:1 to 65:1, 150:1 to 60:1, 150:1 to 55:1, 150:1 to 50:1, 150:1 to 45:1, 150:1 to 40:1, 150:1 to 35:1, 150:1 to 30:1, 150:1 to 25:1, 150:1 to 20:1, 150:1 to 15:1, 150:1 to 10:1, 150:1 to 5:1, 150:1 to 4:1, 150:1 to 3:1, 150:1 to 2:1, 150:1 to 1:1, 140:1 to 85:1, 140:1 to 80:1, 140:1 to 75:1, 140:1 to 70:1, 140:1 to 65:1, 140:1 to

[0224] 60:1, 140:1 to 55:1, 140:1 to 50:1, 140:1 to 45:1, 140:1 to 40:1, 140:1 to 35:1, 140:1 to 30:1,

[0225] 140:1 to 25:1, 140:1 to 20:1, 140:1 to 15:1, 140:1 to 10:1, 140:1 to 5:1, 140:1 to 4:1, 140:1 to 3:1, 140:1 to 2:1, 140:1 to 1:1, 130:1 to 85:1, 130:1 to 80:1, 130:1 to 75:1, 130:1 to 70:1, 130:1 to

[0226] 65:1, 130:1 to 60:1, 130:1 to 55:1, 130:1 to 50:1, 130:1 to 45:1, 130:1 to 40:1, 130:1 to 35:1,

[0227] 130:1 to 30:1, 130:1 to 25:1, 130:1 to 20:1, 130:1 to 15:1, 130:1 to 10:1, 130:1 to 5:1, 130:1 to 4:1, 130:1 to 3:1, 130:1 to 2:1, 130:1 to 1:1, 120:1 to 85:1, 120:1 to 80:1, 120:1 to 75:1, 120:1 to 70:1, 120:1 to 65:1, 120:1 to 60:1, 120:1 to 55:1, 120:1 to 50:1, 120:1 to 45:1, 120:1 to 40:1, 120:1 to 35:1, 120:1 to 30:1, 120:1 to 25:1, 120:1 to 20:1, 120:1 to 15:1, 120:1 to 10:1, 120:1 to

[0228] 5:1, 120:1 to 4:1, 120:1 to 3:1, 120:1 to 2:1, 120:1 to 1:1, 110:1 to 85:1, 110:1 to 80:1, 110:1 to

[0229] 75:1, 110:1 to 70:1, 110:1 to 65:1, 110:1 to 60:1, 110:1 to 55:1, 110:1 to 50:1, 110:1 to 45:1, 110:1 to 40:1, 110:1 to 35:1, 110:1 to 30:1, 110:1 to 25:1, 110:1 to 20:1, 110:1 to 15:1, 110:1 to

[0230] 10:1, 110:1 to 5:1, 110:1 to 4:1, 110:1 to 3:1, 110:1 to 2:1, 110:1 to 1:1, 100:1 to 85:1, 100:1 to

[0231] 80:1, 100:1 to 75:1, 100:1 to 70:1, 100:1 to 65:1, 100:1 to 60:1, 100:1 to 55:1, 100:1 to 50:1, 100:1 to 45:1, 100:1 to 40:1, 100:1 to 35:1, 100:1 to 30:1, 100:1 to 25:1, 100:1 to 20:1, 100:1 to 15:1, 100:1 to 10:1, 100:1 to 5:1, 100:1 to 4:1, 100:1 to 3:1, 100:1 to 2:1, 100:1 to 1:1, 90:1 to 85:1, 90:1 to 80:1, 90:1 to 75:1, 90:1 to 70:1, 90:1 to 65:1, 90:1 to 60:1, 90:1 to 55:1, 90:1 to 50:1,

[0232] 90:1 to 45:1, 90:1 to 40:1, 90:1 to 35:1, 90:1 to 30:1, 90:1 to 25:1, 90:1 to 20:1, 90:1 to 15:1, 90:1 to 10:1, 90:1 to 5:1, 90:1 to 4:1, 90:1 to 3:1, 90:1 to 2:1, 90:1 to 1:1, 80:1 to 60:1, 80:1 to 55:1,

[0233] 80:1 to 50:1, 80:1 to 45:1, 80:1 to 40:1, 80:1 to 35:1, 80:1 to 30:1, 80:1 to 25:1, 80:1 to 20:1, 80:1 to 15:1, 80:1 to 10:1, 80:1 to 5:1, 80:1 to 4:1, 80:1 to 3:1, 80:1 to 2:1, 80:1 to 1:1, 70:1 to 60:1, 70:1 to 55:1, 70:1 to 50:1, 70:1 to 45:1, 70:1 to 40:1, 70:1 to 35:1, 70:1 to 30:1, 70:1 to 25:1, 70:1 to 20:1, 70:1 to 15:1, 70:1 to 10:1, 70:1 to 5:1, 70:1 to 4:1, 70:1 to 3:1, 70:1 to 2:1, 70:1 to 1:1, or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0234] Molar ratio of potassium ions to sodium ions

[0235] In some embodiments, before lyophilization, the molar ratio of the quantity of K+ions (measured in moles) to the quantity of Na+ions (measured in moles) in the lyophilization solution is at least or about 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550: 1 , 500: 1 , 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, 0.1:1, or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 5:1, 950:1 to 5:1, 900:1 to 5:1, 850:1 to 5:1, 800:1 to 5:1, 750:1 to 5:1, 700:1 to 5:1, 650:1 to 5:1, 600:1 to 5:1, 550:1 to 5:1, 500:1 to 5:1, 450:1 to 5:1, 400:1 to 5:1, 350:1 to 5:1, 300:1 to 5:1, 250:1 to 5:1, 200:1 to 5:1, 150:1 to 5:1, 140:1 to 5:1, 130:1 to 5:1, 125:1 to 5:1, 120:1 to 5:1, 115:1 to 5:1, 110:1 to 5:1, 105:1 to 5:1, 100:1 to 5:1, 95:1 to 5:1, 90:1 to 5:1, 85:1 to 5:1, 80:1 to 5:1, 75:1 to 5:1, 70:1 to 5:1, 65:1 to 5:1, 60:1 to 5:1, 55:1 to 5:1, 50:1 to 5:1, 45:1 to 5:1, 40:1 to 5:1, 35:1 to 5:1, 30:1 to 5:1, 25:1 to 5:1, 20:1 to 5:1, 15:1 to 5:1, 10:1 to 5:1, 1000:1 to 10:1, 950:1 to 10:1, 900:1 to 10:1, 850:1 to 10:1, 800:1 to 10:1,

[0236] 750:1 to 10:1, 700:1 to 10:1, 650:1 to 10:1, 600:1 to 10:1, 550:1 to 10:1, 500:1 to 10:1, 450:1 to 10:1, 400:1 to 10:1, 350:1 to 10:1, 300:1 to 10:1, 250:1 to 10:1, 200:1 to 10:1, 150:1 to 10:1, 140:1 to 10:1, 130:1 to 10:1, 125:1 to 10:1, 120:1 to 10:1, 115:1 to 10:1, 110:1 to 10:1, 105:1 to 10:1, 100:1 to 10:1, 95:1 to 10:1, 90:1 to 10:1, 85:1 to 10:1, 80:1 to 10:1, 75:1 to 10:1, 70:1 to 10:1, 65:1 to 10:1, 60:1 to 10:1, 55:1 to 10:1, 50:1 to 10:1, 45:1 to 10:1, 40:1 to 10:1, 35:1 to 10:1, 30:1 to 10:1, 25:1 to 10:1, 20:1 to 10:1, 15:1 to 10:1, 1000:1 to 20:1, 950:1 to 20:1, 900:1 to 20:1, 850: 1 to 20: 1 , 800: 1 to 20: 1 , 750: 1 to 20: 1 , 700: 1 to 20: 1 , 650: 1 to 20: 1 , 600: 1 to 20: 1 , 550: 1 to 20:1, 500:1 to 20:1, 450:1 to 20:1, 400:1 to 20:1, 350:1 to 20:1, 300:1 to 20:1, 250:1 to 20:1, 200:1 to 20:1, 150:1 to 20:1, 140:1 to 20:1, 130:1 to 20:1, 125:1 to 20:1, 120:1 to 20:1, 115:1 to 20:1, 110:1 to 20:1, 105:1 to 20:1, 100:1 to 20:1, 95:1 to 20:1, 90:1 to 20:1, 85:1 to 20:1, 80:1 to 20:1, 75:1 to 20:1, 70:1 to 20:1, 65:1 to 20:1, 60:1 to 20:1, 55:1 to 20:1, 50:1 to 20:1, 45:1 to 20:1, 40:1 to 20:1, 35:1 to 20:1, 30:1 to 20:1, 25:1 to 20:1, 1000:1 to 50:1, 950:1 to 50:1, 900:1 to 50:1, 850:1 to 50:1, 800:1 to 50:1, 750:1 to 50:1, 700:1 to 50:1, 650:1 to 50:1, 600:1 to 50:1, 550:1 to 50:1, 500:1 to 50:1, 450:1 to 50:1, 400:1 to 50:1, 350:1 to 50:1, 300:1 to 50:1, 250:1 to 50:1, 200:1 to 50:1, 150:1 to 50:1, 140:1 to 50:1, 130:1 to 50:1, 125:1 to 50:1, 120:1 to 50:1, 115:1 to 50:1, 110:1 to 50:1, 105:1 to 50:1, 100:1 to 50:1, 95:1 to 50:1, 90:1 to 50:1, 85:1 to 50:1, 80:1 to 50: 1 , 75: 1 to 50: 1 , 70: 1 to 50: 1 , 65: 1 to 50: 1 , 60: 1 to 50: 1 , 55: 1 to 50: 1 , or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein, if present, can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0237] Sucrose concentration

[0238] In some embodiments, before lyophilization, the lyophilization solution further comprises sucrose. In some embodiments, before lyophilization, the lyophilization solution comprises at least or about 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, or 15.0% sucrose on a weight by weight (w / w) or weight by volume (w / v) basis, or concentration between or range encompassing any of the foregoing specifically enumerated concentrations as end points, such as 0.5%-15.0%, 1.0%-14.5%, 1.5%-2.0%, 1.5%- 2.5%, 1.5%-3.0%, 1.5%-3.5%, 1.5%-4.0%, 1.5%-4.5%, 1.5%-5.0%, 1.5%-5.5%, 1.5%-6.0%, 1.5%-6.5%, 1.5%-7.0%, 1.5%-7.5%, 1.5%-8.0%, 1.5%-8.5%, 1.5%-9.0%, 1.5%-9.5%, 1.5%- 10.0%, 1.5%-10.5%, 1.5%-11.0%, 1.5%-11.5%, 1.5%-12.0%, 1.5%-12.5%, 1.5%-13.0%, 1.5%- 13.5%, 1.5%-14.0%, 4.0%-5.0%, 3.5%-5.5%, 3.0%-6.0%, 2.5%-6.5%, or 2.0%-7.0%, or some other concentration or range.

[0239] In some embodiments, before lyophilization, the lyophilization solution comprises carrier protein and a mass of dissolved sucrose that is at least or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,

[0240] 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, or 25.0 times the mass of carrier protein in the lyophilization solution, or multiple between or range encompassing any of the foregoing specifically enumerated multiples as end points, such as 1.0 to 25.0 times, 2.0 to 24.0 times, 2.5 to 23.0 times, 2.5 to 22.0 times, 2.5 to 21.0 times, 2.5 to 20.0 times, 2.5 to 19.0 times, 2.5 to 18.0 times, 2.5 to 17.0 times, 2.5 to 16.0 times, 2.5 to 15.0 times, 2.5 to 14.0 times, 2.5 to 13.0 times,

[0241] 2.5 to 12.0 times, 2.5 to 11.0 times, 2.5 to 10.0 times, 3.0 to 23.0 times, 3.0 to 22.0 times, 3.0 to 21.0 times, 3.0 to 20.0 times, 3.0 to 19.0 times, 3.0 to 18.0 times, 3.0 to 17.0 times, 3.0 to 16.0 times, 3.0 to 15.0 times, 3.0 to 14.0 times, 3.0 to 13.0 times, 3.0 to 12.0 times, 3.0 to 11.0 times, 3.0 to 10.0 times, 3.5 to 23.0 times, 3.5 to 22.0 times, 3.5 to 21.0 times, 3.5 to 20.0 times, 3.5 to 19.0 times, 3.5 to 18.0 times, 3.5 to 17.0 times, 3.5 to 16.0 times, 3.5 to 15.0 times, 3.5 to 14.0 times, 3.5 to 13.0 times, 3.5 to 12.0 times, 3.5 to 11 .0 times, 3.5 to 10.0 times, 4.0 to 23.0 times, 4.0 to 22.0 times, 4.0 to 21.0 times, 4.0 to 20.0 times, 4.0 to 19.0 times, 4.0 to 18.0 times, 4.0 to 17.0 times, 4.0 to 16.0 times, 4.0 to 15.0 times, 4.0 to 14.0 times, 4.0 to 13.0 times, 4.0 to 12.0 times, 4.0 to 11.0 times, 4.0 to 10.0 times, 4.5 to 23.0 times, 4.5 to 22.0 times, 4.5 to 21.0 times,

[0242] 4.5 to 20.0 times, 4.5 to 19.0 times, 4.5 to 18.0 times, 4.5 to 17.0 times, 4.5 to 16.0 times, 4.5 to 15.0 times, 4.5 to 14.0 times, 4.5 to 13.0 times, 4.5 to 12.0 times, 4.5 to 11.0 times, 4.5 to 10.0 times, 5.0 to 23.0 times, 5.0 to 22.0 times, 5.0 to 21 .0 times, 5.0 to 20.0 times, 5.0 to 19.0 times, 5.0 to 18.0 times, 5.0 to 17.0 times, 5.0 to 16.0 times, 5.0 to 15.0 times, 5.0 to 14.0 times, 5.0 to 13.0 times, 5.0 to 12.0 times, 5.0 to 11.0 times, 5.0 to 10.0 times, 5.5 to 23.0 times, 5.5 to 22.0 times, 5.5 to 21.0 times, 5.5 to 20.0 times, 5.5 to 19.0 times, 5.5 to 18.0 times, 5.5 to 17.0 times,

[0243] 5.5 to 16.0 times, 5.5 to 15.0 times, 5.5 to 14.0 times, 5.5 to 13.0 times, 5.5 to 12.0 times, 5.5 to 11.0 times, 5.5 to 10.0 times, 6.0 to 23.0 times, 6.0 to 22.0 times, 6.0 to 21.0 times, 6.0 to 20.0 times, 6.0 to 19.0 times, 6.0 to 18.0 times, 6.0 to 17.0 times, 6.0 to 16.0 times, 6.0 to 15.0 times, 6.0 to 14.0 times, 6.0 to 13.0 times, 6.0 to 12.0 times, 6.0 to 11.0 times, 6.0 to 10.0 times, 6.5 to 23.0 times, 6.5 to 22.0 times, 6.5 to 21.0 times, 6.5 to 20.0 times, 6.5 to 19.0 times, 6.5 to 18.0 times, 6.5 to 17.0 times, 6.5 to 16.0 times, 6.5 to 15.0 times, 6.5 to 14.0 times, 6.5 to 13.0 times,

[0244] 6.5 to 12.0 times, 6.5 to 11.0 times, 6.5 to 10.0 times, 7.0 to 23.0 times, 7.0 to 22.0 times, 7.0 to 21.0 times, 7.0 to 20.0 times, 7.0 to 19.0 times, 7.0 to 18.0 times, 7.0 to 17.0 times, 7.0 to 16.0 times, 7.0 to 15.0 times, 7.0 to 14.0 times, 7.0 to 13.0 times, 7.0 to 12.0 times, 7.0 to 11.0 times, 7.0 to 10.0 times, 7.4 to 7.6 times, 7.3 to 7.7 times, 7.2 to 7.8 times, 7.1 to 7.9 times, 7.0 to 8.0 times, 6.9 to 8.1 times, 6.8 to 8.2 times, 6.7 to 8.3 times, 6.6 to 8.4 times, 6.5 to 8.5 times, 6.4 to

[0245] 8.6 times, 6.3 to 8.7 times, 6.2 to 8.8 times, 6.1 to 8.9 times, 6.0 to 9.0 times, 5.9 to 9.1 times, 5.8 to 9.2 times, 5.7 to 9.3 times, 5.6 to 9.4 times, 5.5 to 9.5 times, 5.4 to 9.6 times, 5.3 to 9.7 times, 5.2 to 9.8 times, 5.1 to 9.9 times, 5.0 to 10.0 times, or some other multiple or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0246] Carrier protein concentration

[0247] In some embodiments, before lyophilization, the lyophilization solution can comprise at least or about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0 grams per liter (g / L) of carrier protein or a concentration between or range encompassing any of the foregoing specifically enumerated concentration values as end points, such as 0.5-12.0, 1.0-11.0, 2.0-10.0, 3.0-9.0, 4.0-8.0, 4.5-7.5, 5.0-7.0, or 5.5-6.5 g / L, or some other concentration or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0248] Certain additional non-limiting embodiments

[0249] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0250] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0251] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0252] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0253] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0254] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0255] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0256] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0257] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and at least 5 mM K+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0258] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0259] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0260] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0261] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0262] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0263] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0264] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0265] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0266] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0267] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0268] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0269] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0270] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0271] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0272] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0273] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0274] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0275] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0276] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0277] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0278] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0279] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0280] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0281] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0282] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0283] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0284] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0285] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0286] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0287] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0288] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0289] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0290] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0291] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0292] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0293] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0294] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0295] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0296] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0297] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0298] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0299] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0300] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0301] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0302] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0303] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0304] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0305] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0306] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0307] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0308] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0309] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0310] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0311] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0312] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0313] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0314] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0315] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0316] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0317] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0318] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0319] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0320] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0321] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0322] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0323] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0324] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0325] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0326] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0327] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0328] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0329] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0330] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0331] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0332] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0333] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0334] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0335] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0336] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0337] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0338] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose at least 2.5 times the amount of CRM197 protein.

[0339] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0340] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0341] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0342] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0343] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0344] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0345] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0346] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0347] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0348] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0349] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0350] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0351] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0352] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0353] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0354] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and at least 5 mM K+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0355] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0356] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0357] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0358] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0359] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0360] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0361] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0362] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0363] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0364] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0365] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0366] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0367] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0368] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0369] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0370] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0371] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0372] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0373] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0374] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0375] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0376] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0377] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0378] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0379] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0380] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0381] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0382] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0383] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0384] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0385] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0386] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0387] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0388] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0389] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0390] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0391] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0392] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0393] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0394] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0395] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0396] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0397] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0398] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0399] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0400] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0401] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0402] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0403] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0404] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0405] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0406] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0407] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0408] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0409] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0410] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0411] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0412] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0413] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0414] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and about 8.5 mM K+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0415] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0416] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0417] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0418] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0419] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0420] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0421] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0422] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0423] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0424] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0425] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0426] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0427] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 2.5 to 10 times the amount of CRM197 protein.

[0428] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0429] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0430] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0431] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0432] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0433] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0434] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0435] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0436] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0437] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0438] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0439] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0440] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0441] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0442] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0443] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0444] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0445] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0446] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0447] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0448] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0449] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0450] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0451] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0452] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0453] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0454] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0455] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0456] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0457] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0458] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0459] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0460] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0461] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0462] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0463] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0464] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0465] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0466] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0467] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0468] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0469] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0470] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0471] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0472] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0473] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0474] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0475] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0476] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0477] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0478] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0479] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0480] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0481] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0482] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0483] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0484] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0485] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0486] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0487] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0488] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0489] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0490] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0491] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0492] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0493] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0494] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0495] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0496] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0497] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0498] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0499] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0500] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0501] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0502] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0503] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0504] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0505] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0506] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0507] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0508] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0509] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0510] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0511] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and about 8.5 mM K+ions. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0512] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0513] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0514] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0515] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0516] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0517] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose 5 to 10 times the amount of CRM197 protein.

[0518] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0519] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0520] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0521] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0522] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0523] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0524] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0525] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0526] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0527] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0528] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0529] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0530] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0531] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0532] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0533] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0534] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0535] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0536] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0537] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0538] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0539] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0540] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and at least 5 mM K+ions.

[0541] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and less than 25 mM Na+ions.

[0542] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8.

[0543] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0544] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0545] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, at least 5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0546] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0547] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration at least 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0548] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0549] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0550] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0551] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0552] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0553] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0554] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0555] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0556] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0557] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0558] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0559] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0560] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0561] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0562] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0563] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0564] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions.

[0565] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0566] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0567] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0568] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0569] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0570] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0571] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and 5 mM to 40 mM K+ions.

[0572] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and less than 25 mM Na+ions. In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8.

[0573] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0574] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0575] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, 5 mM to 40 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0576] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0577] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration 5 mM to 40 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0578] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0579] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0580] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0581] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0582] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0583] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein. In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0584] In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0585] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0586] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0587] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0588] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0589] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0590] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0591] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0592] In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0593] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0594] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0595] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8. In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0596] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0597] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0598] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0599] In some embodiments, before lyophilization, the lyophilization solution comprises 5-7 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0600] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein and about 8.5 mM K+ions.

[0601] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and less than 25 mM Na+ions.

[0602] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8.

[0603] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and less than 25 mM Na+ions.

[0604] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0605] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein, about 8.5 mM K+ions, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0606] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8 and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0607] In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g / L of CRM197 carrier protein in a potassium phosphate buffer of concentration about 5 mM and pH 7.0 to pH 7.8, less than 25 mM Na+ions and a mass of dissolved sucrose about 7.5 times the amount of CRM197 protein.

[0608] Lyophilizate

[0609] Various non-limiting embodiments of the lyophilizate formed following lyophilization of the lyophilization solution comprising carrier protein are described further below. In some embodiments, lyophilizate comprises potassium ions in specified molar ratio relative to the amount of carrier protein. In some embodiments, lyophilizate comprises potassium ions in specified molar ratio relative to an amount of sodium ions which are also present. In some embodiments, lyophilizate comprises sucrose in specified mass ratio relative to the amount of carrier protein. In any of these embodiments, the carrier protein can be a CRM protein, including but not limited to CRM197 protein and variants thereof.

[0610] Molar ratio of potassium ions to carrier protein

[0611] In some embodiments, the molar ratio of the quantity of potassium (measured in moles) to the quantity of carrier protein (measured in moles) in the lyophilizate formed after lyophilization is complete is at least or about 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30: 1 , 25: 1 , 20: 1 , 15:1, 10:1,5:1,4:1,3:1,2:1, 1 : 1 , or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 1:1, 900:1 to 2:1, 800:1 to 3:1, 700:1 to 4:1, 600:1 to 5:1, 500:1 to 10:1, 400:1 to 15:1, 300:1 to 85:1, 300: 1 to 80: 1 , 300: 1 to 75: 1 , 300: 1 to 70: 1 , 300: 1 to 65: 1 , 300: 1 to 60: 1 , 300: 1 to 55: 1 , 300: 1 to 50:1, 300:1 to 45:1, 300:1 to 40:1, 300:1 to 35:1, 300:1 to 30:1, 300:1 to 25:1, 300:1 to 20:1, 300:1 to 15:1, 300:1 to 10:1, 300:1 to 5:1, 300:1 to 4:1, 300:1 to 3:1, 300:1 to 2:1, 300:1 to 1:1, 200: 1 to 85: 1 , 200: 1 to 80: 1 , 200: 1 to 75: 1 , 200: 1 to 70: 1 , 200: 1 to 65: 1 , 200: 1 to 60: 1 , 200: 1 to 55:1, 200:1 to 50:1, 200:1 to 45:1, 200:1 to 40:1, 200:1 to 35:1, 200:1 to 30:1, 200:1 to 25:1, 200:1 to 20:1, 200:1 to 15:1, 200:1 to 10:1, 200:1 to 5:1, 200:1 to 4:1, 200:1 to 3:1, 200:1 to 2:1, 200:1 to 1:1, 150:1 to 85:1, 150:1 to 80:1, 150:1 to 75:1, 150:1 to 70:1, 150:1 to 65:1, 150:1 to 60:1, 150:1 to 55:1, 150:1 to 50:1, 150:1 to 45:1, 150:1 to 40:1, 150:1 to 35:1, 150:1 to 30:1, 150:1 to 25:1, 150:1 to 20:1, 150:1 to 15:1, 150:1 to 10:1, 150:1 to 5:1, 150:1 to4:1, 150:1 to 3:1, 150:1 to 2:1, 150:1 to 1:1, 140:1 to 85:1, 140:1 to 80:1, 140:1 to 75:1, 140:1 to 70:1, 140:1 to 65:1, 140:1 to 60:1, 140:1 to 55:1, 140:1 to 50:1, 140:1 to 45:1, 140:1 to 40:1, 140:1 to 35:1, 140:1 to 30:1, 140:1 to 25:1, 140:1 to 20:1, 140:1 to 15:1, 140:1 to 10:1, 140:1 to 5:1, 140:1 to 4:1, 140:1 to 3:1, 140:1 to 2:1, 140:1 to 1:1, 130:1 to 85:1, 130:1 to 80:1, 130:1 to 75:1, 130:1 to 70:1, 130:1 to 65:1, 130:1 to 60:1, 130:1 to 55:1, 130:1 to 50:1, 130:1 to 45:1, 130:1 to 40:1, 130:1 to 35:1, 130:1 to 30:1, 130:1 to 25:1, 130:1 to 20:1, 130:1 to 15:1, 130:1 to 10:1, 130:1 to 5:1, 130:1 to 4:1, 130:1 to 3:1, 130:1 to 2:1, 130:1 to 1:1, 120:1 to 85:1, 120:1 to 80:1, 120:1 to 75:1, 120:1 to 70:1, 120:1 to 65:1, 120:1 to 60:1, 120:1 to 55:1, 120:1 to 50:1, 120:1 to 45:1, 120:1 to 40:1, 120:1 to 35:1, 120:1 to 30:1, 120:1 to 25:1, 120:1 to 20:1, 120:1 to 15:1, 120:1 to 10:1, 120:1 to 5:1, 120:1 to 4:1, 120:1 to 3:1, 120:1 to 2:1, 120:1 to 1:1, 110:1 to 85:1, 110:1 to 80:1, 110:1 to 75:1, 110:1 to 70:1, 110:1 to 65:1, 110:1 to 60:1, 110:1 to 55:1, 110:1 to 50:1, 110:1 to 45:1, 110:1 to 40:1, 110:1 to 35:1, 110:1 to 30:1, 110:1 to 25:1, 110:1 to 20:1, 110:1 to 15:1, 110:1 to 10:1, 110:1 to 5:1, 110:1 to 4:1, 110:1 to 3:1, 110:1 to 2:1, 110:1 to 1:1, 100:1 to 85:1, 100:1 to 80:1, 100:1 to 75:1, 100:1 to 70:1, 100:1 to 65:1, 100:1 to 60:1, 100:1 to 55:1, 100:1 to 50:1, 100:1 to 45:1, 100:1 to 40:1, 100:1 to 35:1, 100:1 to 30:1, 100:1 to 25:1, 100:1 to 20:1, 100:1 to 15:1, 100:1 to 10:1, 100:1 to 5:1, 100:1 to 4:1, 100:1 to 3:1, 100:1 to 2:1, 100:1 to 1:1, 90:1 to 85:1, 90:1 to 80:1, 90:1 to 75:1, 90:1 to 70:1, 90:1 to 65:1, 90:1 to 60:1, 90:1 to 55:1, 90:1 to 50:1, 90:1 to 45:1, 90:1 to 40:1, 90:1 to 35:1, 90:1 to 30:1, 90:1 to 25:1, 90:1 to 20:1, 90:1 to 15:1, 90:1 to 10:1, 90:1 to 5:1, 90:1 to 4:1, 90:1 to 3:1, 90:1 to 2:1, 90:1 to 1:1, 80:1 to 60:1, 80:1 to 55:1, 80:1 to 50:1, 80:1 to 45:1, 80:1 to 40:1, 80:1 to 35:1, 80:1 to 30:1, 80:1 to 25:1, 80:1 to 20:1, 80:1 to 15:1, 80:1 to 10:1, 80:1 to 5:1, 80:1 to 4:1, 80:1 to 3:1, 80:1 to 2:1, 80:1 to 1:1, 70:1 to 60:1, 70:1 to 55:1, 70:1 to 50:1, 70:1 to 45:1, 70:1 to 40:1, 70:1 to 35:1, 70:1 to 30:1, 70:1 to 25:1, 70:1 to 20:1, 70:1 to 15:1, 70:1 to 10:1, 70:1 to 5:1, 70:1 to 4:1, 70:1 to 3:1, 70:1 to 2:1, 70:1 to 1:1, or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0612] Molar ratio of potassium ions to sodium ions

[0613] In some embodiments, the molar ratio of the quantity of K+ions (measured in moles) to the quantity of Na+ions (measured in moles) in the lyophilizate formed after lyophilization is complete is at least or about 1,000,000:1, 500,000:1, 100,000:1, 50,000:1, 10,000:1, 5000:1, 1000: 1 , 950: 1 , 900: 1 , 850: 1 , 800: 1 , 750: 1 , 700: 1 , 650: 1 , 600: 1 , 550: 1 , 500: 1 , 450: 1 , 400: 1 , 350: 1 , 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, 0.1:1, or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 5:1, 950:1 to 5:1, 900:1 to 5:1, 850:1 to 5:1, 800:1 to 5:1, 750:1 to 5:1, 700:1 to 5:1, 650:1 to 5:1, 600:1 to 5:1, 550:1 to 5:1, 500:1 to 5:1, 450:1 to 5:1, 400:1 to 5:1, 350:1 to 5:1, 300:1 to 5:1, 250:1 to 5:1, 200:1 to 5:1, 150:1 to 5:1, 140:1 to 5:1, 130:1 to 5:1, 125:1 to 5:1, 120:1 to 5:1, 115:1 to 5:1, 110:1 to 5:1, 105:1 to 5:1, 100:1 to 5:1, 95:1 to 5:1, 90:1 to 5:1, 85:1 to 5:1, 80:1 to 5:1, 75:1 to 5:1, 70:1 to 5:1, 65:1 to 5:1, 60:1 to 5:1, 55:1 to 5:1, 50:1 to 5:1, 45:1 to 5:1, 40:1 to 5:1, 35:1 to 5:1, 30:1 to 5:1, 25:1 to 5:1, 20:1 to 5:1, 15:1 to 5:1, 10:1 to 5:1, 1000:1 to 10:1, 950:1 to 10:1, 900:1 to 10:1, 850:1 to 10:1, 800:1 to 10:1, 750:1 to 10:1, 700:1 to 10:1, 650:1 to 10:1, 600:1 to 10:1, 550:1 to 10:1, 500:1 to 10:1, 450:1 to 10:1, 400:1 to 10:1, 350:1 to 10:1, 300:1 to 10:1, 250:1 to 10:1, 200:1 to 10:1, 150:1 to 10:1, 140:1 to 10:1, 130:1 to 10:1, 125:1 to 10:1, 120:1 to 10:1, 115:1 to 10:1, 110:1 to 10:1, 105:1 to 10:1, 100:1 to 10:1, 95:1 to 10:1, 90:1 to 10:1, 85:1 to 10:1, 80:1 to 10:1, 75:1 to 10:1, 70:1 to 10:1, 65:1 to 10:1, 60:1 to 10:1, 55:1 to 10:1, 50:1 to 10:1, 45:1 to 10:1, 40:1 to 10:1, 35:1 to 10:1, 30:1 to 10:1, 25:1 to 10:1, 20:1 to 10:1, 15:1 to 10:1, 1000:1 to 20:1, 950:1 to 20:1, 900:1 to 20:1, 850:1 to 20:1, 800:1 to 20:1, 750: 1 to 20: 1 , 700: 1 to 20: 1 , 650: 1 to 20: 1 , 600: 1 to 20: 1 , 550: 1 to 20: 1 , 500: 1 to 20: 1 , 450: 1 to 20:1, 400:1 to 20:1, 350:1 to 20:1, 300:1 to 20:1, 250:1 to 20:1, 200:1 to 20:1, 150:1 to 20:1, 140:1 to 20:1, 130:1 to 20:1, 125:1 to 20:1, 120:1 to 20:1, 115:1 to 20:1, 110:1 to 20:1, 105:1 to 20:1, 100:1 to 20:1, 95:1 to 20:1, 90:1 to 20:1, 85:1 to 20:1, 80:1 to 20:1, 75:1 to 20:1, 70:1 to 20:1, 65:1 to 20:1, 60:1 to 20:1, 55:1 to 20:1, 50:1 to 20:1, 45:1 to 20:1, 40:1 to 20:1, 35:1 to 20:1, 30:1 to 20:1, 25:1 to 20:1, 1000:1 to 50:1, 950:1 to 50:1, 900:1 to 50:1, 850:1 to 50:1, 800:1 to 50:1, 750:1 to 50:1, 700:1 to 50:1, 650:1 to 50:1, 600:1 to 50:1, 550:1 to 50:1, 500:1 to 50:1, 450:1 to 50:1, 400:1 to 50:1, 350:1 to 50:1, 300:1 to 50:1, 250:1 to 50:1, 200:1 to 50:1, 150:1 to 50:1, 140:1 to 50:1, 130:1 to 50:1, 125:1 to 50:1, 120:1 to 50:1, 115:1 to 50:1, 110:1 to 50:1, 105:1 to 50:1, 100:1 to 50:1, 95:1 to 50:1, 90:1 to 50:1, 85:1 to 50:1, 80:1 to 50:1, 75:1 to 50:1, 70:1 to 50:1, 65:1 to 50:1, 60:1 to 50:1, 55:1 to 50:1, or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein, if present, can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0614] Sucrose concentration

[0615] In some embodiments, the lyophilizate formed after lyophilization is complete comprises carrier protein and a mass of sucrose that is at least or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5,

[0616] 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,

[0617] 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,

[0618] 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,

[0619] 17.5, 18.0, 18.5, 19.0, 19.5, 20.0,21.0, 22.0, 23.0, 24.0, or 25.0 times the mass of carrier protein in the lyophilizate, or multiple between or range encompassing any of the foregoing specifically enumerated multiples as end points, such as 1.0 to 25.0 times, 2.0 to 24.0 times, 2.5 to 23.0 times, 2.5 to 22.0 times, 2.5 to 21.0 times, 2.5 to 20.0 times, 2.5 to 19.0 times, 2.5 to 18.0 times,

[0620] 2.5 to 17.0 times, 2.5 to 16.0 times, 2.5 to 15.0 times, 2.5 to 14.0 times, 2.5 to 13.0 times, 2.5 to 12.0 times, 2.5 to 11.0 times, 2.5 to 10.0 times, 3.0 to 23.0 times, 3.0 to 22.0 times, 3.0 to 21.0 times, 3.0 to 20.0 times, 3.0 to 19.0 times, 3.0 to 18.0 times, 3.0 to 17.0 times, 3.0 to 16.0 times, 3.0 to 15.0 times, 3.0 to 14.0 times, 3.0 to 13.0 times, 3.0 to 12.0 times, 3.0 to 11.0 times, 3.0 to 10.0 times, 3.5 to 23.0 times, 3.5 to 22.0 times, 3.5 to 21.0 times, 3.5 to 20.0 times, 3.5 to 19.0 times, 3.5 to 18.0 times, 3.5 to 17.0 times, 3.5 to 16.0 times, 3.5 to 15.0 times, 3.5 to 14.0 times,

[0621] 3.5 to 13.0 times, 3.5 to 12.0 times, 3.5 to 11.0 times, 3.5 to 10.0 times, 4.0 to 23.0 times, 4.0 to 22.0 times, 4.0 to 21.0 times, 4.0 to 20.0 times, 4.0 to 19.0 times, 4.0 to 18.0 times, 4.0 to 17.0 times, 4.0 to 16.0 times, 4.0 to 15.0 times, 4.0 to 14.0 times, 4.0 to 13.0 times, 4.0 to 12.0 times, 4.0 to 11.0 times, 4.0 to 10.0 times, 4.5 to 23.0 times, 4.5 to 22.0 times, 4.5 to 21.0 times, 4.5 to 20.0 times, 4.5 to 19.0 times, 4.5 to 18.0 times, 4.5 to 17.0 times, 4.5 to 16.0 times, 4.5 to 15.0 times, 4.5 to 14.0 times, 4.5 to 13.0 times, 4.5 to 12.0 times, 4.5 to 11.0 times, 4.5 to 10.0 times, 5.0 to 23.0 times, 5.0 to 22.0 times, 5.0 to 21.0 times, 5.0 to 20.0 times, 5.0 to 19.0 times, 5.0 to 18.0 times, 5.0 to 17.0 times, 5.0 to 16.0 times, 5.0 to 15.0 times, 5.0 to 14.0 times, 5.0 to 13.0 times, 5.0 to 12.0 times, 5.0 to 11.0 times, 5.0 to 10.0 times, 5.5 to 23.0 times, 5.5 to 22.0 times,

[0622] 5.5 to 21.0 times, 5.5 to 20.0 times, 5.5 to 19.0 times, 5.5 to 18.0 times, 5.5 to 17.0 times, 5.5 to 16.0 times, 5.5 to 15.0 times, 5.5 to 14.0 times, 5.5 to 13.0 times, 5.5 to 12.0 times, 5.5 to 11.0 times, 5.5 to 10.0 times, 6.0 to 23.0 times, 6.0 to 22.0 times, 6.0 to 21 .0 times, 6.0 to 20.0 times, 6.0 to 19.0 times, 6.0 to 18.0 times, 6.0 to 17.0 times, 6.0 to 16.0 times, 6.0 to 15.0 times, 6.0 to 14.0 times, 6.0 to 13.0 times, 6.0 to 12.0 times, 6.0 to 11.0 times, 6.0 to 10.0 times, 6.5 to 23.0 times, 6.5 to 22.0 times, 6.5 to 21.0 times, 6.5 to 20.0 times, 6.5 to 19.0 times, 6.5 to 18.0 times,

[0623] 6.5 to 17.0 times, 6.5 to 16.0 times, 6.5 to 15.0 times, 6.5 to 14.0 times, 6.5 to 13.0 times, 6.5 to 12.0 times, 6.5 to 11.0 times, 6.5 to 10.0 times, 7.0 to 23.0 times, 7.0 to 22.0 times, 7.0 to 21.0 times, 7.0 to 20.0 times, 7.0 to 19.0 times, 7.0 to 18.0 times, 7.0 to 17.0 times, 7.0 to 16.0 times, 7.0 to 15.0 times, 7.0 to 14.0 times, 7.0 to 13.0 times, 7.0 to 12.0 times, 7.0 to 11.0 times, 7.0 to 10.0 times, 7.4 to 7.6 times, 7.3 to 7.7 times, 7.2 to 7.8 times, 7.1 to 7.9 times, 7.0 to 8.0 times, 6.9 to 8.1 times, 6.8 to 8.2 times, 6.7 to 8.3 times, 6.6 to 8.4 times, 6.5 to 8.5 times, 6.4 to 8.6 times, 6.3 to 8.7 times, 6.2 to 8.8 times, 6.1 to 8.9 times, 6.0 to 9.0 times, 5.9 to 9.1 times, 5.8 to 9.2 times, 5.7 to 9.3 times, 5.6 to 9.4 times, 5.5 to 9.5 times, 5.4 to 9.6 times, 5.3 to 9.7 times, 5.2 to 9.8 times, 5.1 to 9.9 times, 5.0 to 10.0 times, or some other multiple or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.

[0624] Reconstituted lyophilizate

[0625] As described above, after lyophilization, lyophilizate comprising carrier protein may be reconstituted in aprotic solvent in preparation for conjugating carrier protein with activated saccharide, for example, by reductive amination. In some embodiments, the aprotic solvent comprises, consists essentially of, or consists of dimethylsulphoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA). In some embodiments, the aprotic solvent comprises, consists essentially of, or consists of dimethylsulphoxide (DMSO) which, in some embodiments, can be anhydrous DMSO. Various non-limiting embodiments of reconstituted lyophilizate are described further below. In some embodiments, reconstituted lyophilizate comprises potassium ions in specified molar ratio relative to the amount of carrier protein. In some embodiments, reconstituted lyophilizate comprises carrier protein which is solubilized in aprotic solvent at higher concentrations than would otherwise occur due to the presence of potassium ions. In some embodiments, reconstituted lyophilizate comprises potassium ions in specified molar ratio relative to an amount of sodium ions which are also present. In some embodiments, reconstituted lyophilizate comprises sucrose in specified mass ratio relative to the amount of carrier protein. In any of these embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof, and the aprotic solvent can be DMSO.

[0626] Potassium and sodium ion concentration In some embodiments, the concentration of potassium ions (K+) in the solution formed by reconstituting the lyophilizate with aprotic solvent before carrying out reductive amination is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 millimolar (mM), or value between or range encompassing any of the foregoing specifically enumerated concentration values as end points, such as 0.5-90 mM, 1-85 mM, 2-80 mM, 3-75 mM, 4-90 mM,

[0627] 4-85 mM, 4-80 mM, 4-75 mM, 4-70 mM, 4-65 mM, 4-60 mM, 4-55 mM, 4-50 mM, 4-45 mM, 4-40 mM, 4-35 mM, 4-30 mM, 4-25 mM, 4-20 mM, 4-18 mM, 4-16 mM, 4-14 mM, 4-12 mM, 4-10 mM,

[0628] 5-90 mM, 5-85 mM, 5-80 mM, 5-75 mM, 5-70 mM, 5-65 mM, 5-60 mM, 5-55 mM, 5-50 mM, 5-45 mM, 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 5-18 mM, 5-16 mM, 5-14 mM, 5-12 mM,

[0629] 5-10 mM, 6-90 mM, 6-85 mM, 6-80 mM, 6-75 mM, 6-70 mM, 6-65 mM, 6-60 mM, 6-55 mM, 6-50 mM, 6-45 mM, 6-40 mM, 6-35 mM, 6-30 mM, 6-25 mM, 6-20 mM, 6-18 mM, 6-16 mM, 6-14 mM,

[0630] 6-12 mM, 6-10 mM, 7-90 mM, 7-85 mM, 7-80 mM, 7-75 mM, 7-70 mM, 7-65 mM, 7-60 mM, 7-55 mM, 7-50 mM, 7-45 mM, 7-40 mM, 7-35 mM, 7-30 mM, 7-25 mM, 7-20 mM, 7-18 mM, 7-16 mM,

[0631] 7-14 mM, 7-12 mM, 7-10 mM, 8-90 mM, 8-85 mM, 8-80 mM, 8-75 mM, 8-70 mM, 8-65 mM, 8-60 mM, 8-55 mM, 8-50 mM, 8-45 mM, 8-40 mM, 8-35 mM, 8-30 mM, 8-25 mM, 8-20 mM, 8-18 mM,

[0632] 8-16 mM, 8-14 mM, 8-12 mM, 8-10 mM, 8.5-9.5 mM, 7.5-10.5 mM, 7-11 mM, 6.5-11.5 mM, 5.5- 12.5 mM, 5-13 mM, or 4.5-13.5 mM, or some other range or value. In any of these embodiments, the solution formed by reconstituting the lyophilizate with aprotic solvent before carrying out reductive amination can, at the same time, comprise less than 25 mM of sodium ions (Na+), whatever the source, for example, less than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1, 0.05 mM sodium ions, or lower amounts of sodium ions, such as not more than trace amounts of sodium ions.

[0633] Molar ratio of potassium ions to carrier protein

[0634] In some embodiments, the molar ratio of the quantity of K+ions (measured in moles) to the quantity of carrier protein (measured in moles) in the solution formed by reconstituting the lyophilizate with aprotic solvent before carrying out reductive amination is at least or about 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 1:1, 900:1 to 2:1, 800:1 to 3:1, 700:1 to 4:1, 600:1 to 5:1, 500:1 to 10:1, 400:1 to 15:1, 300:1 to 85:1, 300:1 to 80:1, 300:1 to 75:1, 300: 1 to 70: 1 , 300: 1 to 65: 1 , 300: 1 to 60: 1 , 300: 1 to 55: 1 , 300: 1 to 50: 1 , 300: 1 to 45: 1 , 300: 1 to 40:1, 300:1 to 35:1, 300:1 to 30:1, 300:1 to 25:1, 300:1 to 20:1, 300:1 to 15:1, 300:1 to 10:1, 300:1 to 5:1, 300:1 to 4:1, 300:1 to 3:1, 300:1 to 2:1, 300:1 to 1:1, 200:1 to 85:1, 200:1 to 80:1, 200: 1 to 75: 1 , 200: 1 to 70: 1 , 200: 1 to 65: 1 , 200: 1 to 60: 1 , 200: 1 to 55: 1 , 200: 1 to 50: 1 , 200: 1 to 45:1, 200:1 to 40:1, 200:1 to 35:1, 200:1 to 30:1, 200:1 to 25:1, 200:1 to 20:1, 200:1 to 15:1, 200:1 to 10:1, 200:1 to 5:1, 200:1 to 4:1, 200:1 to 3:1, 200:1 to 2:1, 200:1 to 1:1, 150:1 to 85:1, 150:1 to 80:1, 150:1 to 75:1, 150:1 to 70:1, 150:1 to 65:1, 150:1 to 60:1, 150:1 to 55:1, 150:1 to 50:1, 150:1 to 45:1, 150:1 to 40:1, 150:1 to 35:1, 150:1 to 30:1, 150:1 to 25:1, 150:1 to 20:1, 150:1 to 15:1, 150:1 to 10:1, 150:1 to 5:1, 150:1 to 4:1, 150:1 to 3:1, 150:1 to 2:1, 150:1 to 1:1, 140:1 to 85:1, 140:1 to 80:1, 140:1 to 75:1, 140:1 to 70:1, 140:1 to 65:1, 140:1 to 60:1, 140:1 to 55:1, 140:1 to 50:1, 140:1 to 45:1, 140:1 to 40:1, 140:1 to 35:1, 140:1 to 30:1, 140:1 to 25:1, 140:1 to 20:1, 140:1 to 15:1, 140:1 to 10:1, 140:1 to 5:1, 140:1 to 4:1, 140:1 to 3:1, 140:1 to 2:1, 140:1 to 1:1, 130:1 to 85:1, 130:1 to 80:1, 130:1 to 75:1, 130:1 to 70:1, 130:1 to 65:1, 130:1 to

[0635] 60:1, 130:1 to 55:1, 130:1 to 50:1, 130:1 to 45:1, 130:1 to 40:1, 130:1 to 35:1, 130:1 to 30:1,

[0636] 130:1 to 25:1, 130:1 to 20:1, 130:1 to 15:1, 130:1 to 10:1, 130:1 to 5:1, 130:1 to4:1, 130:1 to 3:1, 130:1 to 2:1, 130:1 to 1:1, 120:1 to 85:1, 120:1 to 80:1, 120:1 to 75:1, 120:1 to 70:1, 120:1 to

[0637] 65:1, 120:1 to 60:1, 120:1 to 55:1, 120:1 to 50:1, 120:1 to 45:1, 120:1 to 40:1, 120:1 to 35:1,

[0638] 120:1 to 30:1, 120:1 to 25:1, 120:1 to 20:1, 120:1 to 15:1, 120:1 to 10:1, 120:1 to 5:1, 120:1 to 4:1, 120:1 to 3:1, 120:1 to 2:1, 120:1 to 1:1, 110:1 to 85:1, 110:1 to 80:1, 110:1 to 75:1, 110:1 to 70:1, 110:1 to 65:1, 110:1 to 60:1, 110:1 to 55:1, 110:1 to 50:1, 110:1 to 45:1, 110:1 to 40:1, 110:1 to 35:1, 110:1 to 30:1, 110:1 to 25:1, 110:1 to 20:1, 110:1 to 15:1, 110:1 to 10:1, 110:1 to 5:1, 110:1 to 4:1, 110:1 to 3:1, 110:1 to 2:1, 110:1 to 1:1, 100:1 to 85:1, 100:1 to 80:1, 100:1 to 75:1, 100:1 to 70:1, 100:1 to 65:1, 100:1 to 60:1, 100:1 to 55:1, 100:1 to 50:1, 100:1 to 45:1, 100:1 to 40:1, 100:1 to 35:1, 100:1 to 30:1, 100:1 to 25:1, 100:1 to 20:1, 100:1 to 15:1, 100:1 to 10:1, 100:1 to 5:1, 100:1 to4:1, 100:1 to 3:1, 100:1 to 2:1, 100:1 to 1:1, 90:1 to 85:1, 90:1 to 80:1, 90:1 to 75:1, 90:1 to 70:1, 90:1 to 65:1, 90:1 to 60:1, 90:1 to 55:1, 90:1 to 50:1, 90:1 to 45:1, 90:1 to 40:1, 90:1 to 35:1, 90:1 to 30:1, 90:1 to 25:1, 90:1 to 20:1, 90:1 to 15:1, 90:1 to 10:1, 90:1 to 5:1, 90:1 to 4:1, 90:1 to 3:1, 90:1 to 2:1, 90:1 to 1:1, 80:1 to 60:1, 80:1 to 55:1, 80:1 to 50:1, 80:1 to 45:1, 80:1 to 40:1, 80:1 to 35:1, 80:1 to 30:1, 80:1 to 25:1, 80:1 to 20:1, 80:1 to 15:1, 80:1 to 10:1, 80:1 to 5:1, 80:1 to 4:1, 80:1 to 3:1, 80:1 to 2:1, 80:1 to 1:1, 70:1 to 60:1, 70:1 to 55:1, 70:1 to 50:1, 70:1 to 45:1, 70:1 to 40:1, 70:1 to 35:1, 70:1 to 30:1, 70:1 to 25:1, 70:1 to 20:1, 70:1 to 15:1, 70:1 to 10:1, 70:1 to 5:1, 70:1 to 4:1, 70:1 to 3:1, 70:1 to 2:1, 70:1 to 1:1, or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof, and the aprotic solvent can be DMSO (including anhydrous DMSO).

[0639] Carrier protein solubility

[0640] In some embodiments, lyophilization of carrier protein in the presence of K+ions as described herein enhances the solubility of the carrier protein in an aprotic solvent, such as DMSO, such that the solubility of lyophilized carrier protein is at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 grams per liter (g / L), or greater, when reconstituted in an aprotic solvent, such as DMSO, or a value between or range encompassing any of the foregoing specifically enumerated values as end points, such as 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-10, 3-9, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 5-25, 5-24,

[0641] 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 6-25, 6-24,

[0642] 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 7-25, 7-24,

[0643] 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 8-25, 8-24,

[0644] 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-25, 9-24,

[0645] 9-23, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-25, 10-24,

[0646] 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11- 25, 11-24, 11-23, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13- 25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-25, 15-24, 15-23, 15-22, 15- 21, 15-20, 15-19, 15-18, 15-17, 15-16, 16-25, 16-24, 16-23, 16-22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-25, 18-24, 18-23, 18-22, 18- 21, 18-20, 18-19, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, 24-25 g / L solubility. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof, the aprotic solvent can be DMSO (including anhydrous DMSO), and the molar ratio of the quantity of K+ions (measured in moles) to the quantity of carrier protein (measured in moles) in the lyophilizate prior to reconstitution in solvent is at least or about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 75:1, 80:1, 90: 1 , 100: 1 , 125: 1 , 150: 1 , 200: 1 , 250: 1 , 300: 1 , 350: 1 , 400: 1 , 450: 1 , or 500: 1 , or a value between or range encompassing any of the foregoing specifically enumerated values as end points, such as 25:1 to 500:1, 50:1 to 500:1, 100:1 to 500:1, 25:1 to 400:1, 50:1 to 400:1, 100:1 to 400:1, 25:1 to 300:1, 50:1 to 300:1, 100:1 to 300:1, 25:1 to 200:1, 50:1 to 200:1, 100:1 to 200:1, 25:1 to 150:1, 25:1 to 125:1, 25:1 to 100:1, 30:1 to 150:1, 30:1 to 125:1, 30:1 to 100:1, 40:1 to 150:1, 40:1 to 125:1, 40:1 to 100:1, 50:1 to 150:1, 50:1 to 125:1, 50:1 to 100:1, 60:1 to 150:1, 60:1 to 125:1, 60:1 to 100:1, 70:1 to 150:1, 70:1 to 125:1, 70:1 to 100:1, 75:1 to 150:1, 75:1 to 125:1, 75:1 to 100:1, 80:1 to 150:1, 80:1 to 125:1, 80:1 to 100:1, or some other molar ratio or range.

[0647] Molar ratio of potassium ions to sodium ions

[0648] In some embodiments, the molar ratio of the quantity of K+ions (measured in moles) to the quantity of Na+ions (measured in moles) in the solution formed by reconstituting the lyophilizate with aprotic solvent before carrying out reductive amination is at least or about 1000: 1 , 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 140:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1, 0.1:1, or molar ratio between or range encompassing any of the foregoing specifically enumerated molar ratios as end points, such as 1000:1 to 5:1, 950:1 to 5:1, 900:1 to 5:1, 850:1 to 5:1, 800:1 to 5:1, 750:1 to 5:1, 700:1 to 5:1, 650:1 to 5:1, 600:1 to 5:1, 550:1 to 5:1, 500:1 to 5:1, 450:1 to 5:1, 400:1 to 5:1, 350:1 to 5:1, 300:1 to 5:1, 250:1 to 5:1, 200:1 to 5:1, 150:1 to 5:1, 140:1 to 5:1, 130:1 to 5:1, 125:1 to 5:1, 120:1 to 5:1, 115:1 to 5:1, 110:1 to 5:1, 105:1 to 5:1, 100:1 to 5:1, 95:1 to 5:1, 90:1 to 5:1, 85:1 to 5:1, 80:1 to 5:1, 75:1 to 5:1, 70:1 to 5:1, 65:1 to 5:1, 60:1 to 5:1, 55:1 to 5:1, 50:1 to 5:1, 45:1 to 5:1, 40:1 to 5:1, 35:1 to 5:1, 30:1 to 5:1, 25:1 to 5:1, 20:1 to 5:1, 15:1 to 5:1, 10:1 to 5:1, 1000:1 to 10:1, 950:1 to 10:1, 900:1 to 10:1, 850:1 to 10:1, 800:1 to 10:1, 750:1 to 10:1, 700:1 to 10:1, 650:1 to 10:1, 600:1 to 10:1, 550:1 to 10:1, 500:1 to 10:1, 450:1 to 10:1, 400:1 to 10:1, 350:1 to 10:1, 300:1 to 10:1, 250:1 to 10:1, 200:1 to 10:1, 150:1 to 10:1, 140:1 to 10:1, 130:1 to 10:1, 125:1 to 10:1, 120:1 to 10:1, 115:1 to 10:1, 110:1 to 10:1, 105:1 to 10:1, 100:1 to 10:1, 95:1 to 10:1, 90:1 to 10:1, 85:1 to 10:1, 80:1 to 10:1, 75:1 to 10:1, 70:1 to 10:1, 65:1 to 10:1, 60:1 to 10:1, 55:1 to 10:1, 50:1 to 10:1, 45:1 to 10:1, 40:1 to 10:1, 35:1 to 10:1, 30:1 to 10:1, 25:1 to 10:1, 20:1 to 10:1, 15:1 to 10:1, 1000:1 to 20:1, 950:1 to 20:1, 900:1 to 20:1, 850:1 to 20:1, 800:1 to 20:1, 750: 1 to 20: 1 , 700: 1 to 20: 1 , 650: 1 to 20: 1 , 600: 1 to 20: 1 , 550: 1 to 20: 1 , 500: 1 to 20: 1 , 450: 1 to 20:1, 400:1 to 20:1, 350:1 to 20:1, 300:1 to 20:1, 250:1 to 20:1, 200:1 to 20:1, 150:1 to 20:1, 140:1 to 20:1, 130:1 to 20:1, 125:1 to 20:1, 120:1 to 20:1, 115:1 to 20:1, 110:1 to 20:1, 105:1 to 20:1, 100:1 to 20:1, 95:1 to 20:1, 90:1 to 20:1, 85:1 to 20:1, 80:1 to 20:1, 75:1 to 20:1, 70:1 to 20:1, 65:1 to 20:1, 60:1 to 20:1, 55:1 to 20:1, 50:1 to 20:1, 45:1 to 20:1, 40:1 to 20:1, 35:1 to 20:1, 30:1 to 20:1, 25:1 to 20:1, 1000:1 to 50:1, 950:1 to 50:1, 900:1 to 50:1, 850:1 to 50:1, 800:1 to 50:1, 750:1 to 50:1, 700:1 to 50:1, 650:1 to 50:1, 600:1 to 50:1, 550:1 to 50:1, 500:1 to 50:1, 450:1 to 50:1, 400:1 to 50:1, 350:1 to 50:1, 300:1 to 50:1, 250:1 to 50:1, 200:1 to 50:1, 150:1 to 50:1, 140:1 to 50:1, 130:1 to 50:1, 125:1 to 50:1, 120:1 to 50:1, 115:1 to 50:1, 110:1 to 50:1, 105:1 to 50:1, 100:1 to 50:1, 95:1 to 50:1, 90:1 to 50:1, 85:1 to 50:1, 80:1 to 50:1, 75:1 to 50:1, 70:1 to 50:1, 65:1 to 50:1, 60:1 to 50:1, 55:1 to 50:1, or some other molar ratio or range. In any of the foregoing embodiments, the carrier protein, if present, can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof, and the solvent can be an aprotic solvent, including but not limited to DMSO (including anhydrous DMSO).

[0649] Sucrose concentration

[0650] In some embodiments, the solution formed by reconstituting the lyophilizate with solvent before carrying out reductive amination comprises carrier protein and a mass of dissolved sucrose that is at least or about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, or 25.0 times the mass of carrier protein in the reconstituted lyophilizate solution, or multiple between or range encompassing any of the foregoing specifically enumerated multiples as end points, such as 1.0 to 25.0 times, 2.0 to 24.0 times, 2.5 to 23.0 times, 2.5 to 22.0 times, 2.5 to 21.0 times, 2.5 to 20.0 times, 2.5 to 19.0 times, 2.5 to 18.0 times, 2.5 to 17.0 times, 2.5 to 16.0 times, 2.5 to 15.0 times, 2.5 to 14.0 times, 2.5 to 13.0 times, 2.5 to 12.0 times, 2.5 to 11.0 times, 2.5 to 10.0 times, 3.0 to 23.0 times, 3.0 to 22.0 times, 3.0 to 21.0 times, 3.0 to 20.0 times, 3.0 to 19.0 times, 3.0 to 18.0 times, 3.0 to 17.0 times, 3.0 to 16.0 times, 3.0 to 15.0 times, 3.0 to 14.0 times, 3.0 to 13.0 times, 3.0 to 12.0 times, 3.0 to 11.0 times, 3.0 to 10.0 times, 3.5 to 23.0 times, 3.5 to 22.0 times, 3.5 to 21.0 times, 3.5 to 20.0 times, 3.5 to 19.0 times, 3.5 to 18.0 times, 3.5 to 17.0 times, 3.5 to 16.0 times, 3.5 to 15.0 times, 3.5 to 14.0 times,

[0651] 3.5 to 13.0 times, 3.5 to 12.0 times, 3.5 to 11.0 times, 3.5 to 10.0 times, 4.0 to 23.0 times, 4.0 to 22.0 times, 4.0 to 21.0 times, 4.0 to 20.0 times, 4.0 to 19.0 times, 4.0 to 18.0 times, 4.0 to 17.0 times, 4.0 to 16.0 times, 4.0 to 15.0 times, 4.0 to 14.0 times, 4.0 to 13.0 times, 4.0 to 12.0 times, 4.0 to 11.0 times, 4.0 to 10.0 times, 4.5 to 23.0 times, 4.5 to 22.0 times, 4.5 to 21.0 times, 4.5 to 20.0 times, 4.5 to 19.0 times, 4.5 to 18.0 times, 4.5 to 17.0 times, 4.5 to 16.0 times, 4.5 to 15.0 times, 4.5 to 14.0 times, 4.5 to 13.0 times, 4.5 to 12.0 times, 4.5 to 11.0 times, 4.5 to 10.0 times, 5.0 to 23.0 times, 5.0 to 22.0 times, 5.0 to 21.0 times, 5.0 to 20.0 times, 5.0 to 19.0 times, 5.0 to 18.0 times, 5.0 to 17.0 times, 5.0 to 16.0 times, 5.0 to 15.0 times, 5.0 to 14.0 times, 5.0 to 13.0 times, 5.0 to 12.0 times, 5.0 to 11.0 times, 5.0 to 10.0 times, 5.5 to 23.0 times, 5.5 to 22.0 times,

[0652] 5.5 to 21.0 times, 5.5 to 20.0 times, 5.5 to 19.0 times, 5.5 to 18.0 times, 5.5 to 17.0 times, 5.5 to 16.0 times, 5.5 to 15.0 times, 5.5 to 14.0 times, 5.5 to 13.0 times, 5.5 to 12.0 times, 5.5 to 11.0 times, 5.5 to 10.0 times, 6.0 to 23.0 times, 6.0 to 22.0 times, 6.0 to 21 .0 times, 6.0 to 20.0 times, 6.0 to 19.0 times, 6.0 to 18.0 times, 6.0 to 17.0 times, 6.0 to 16.0 times, 6.0 to 15.0 times, 6.0 to 14.0 times, 6.0 to 13.0 times, 6.0 to 12.0 times, 6.0 to 11.0 times, 6.0 to 10.0 times, 6.5 to 23.0 times, 6.5 to 22.0 times, 6.5 to 21.0 times, 6.5 to 20.0 times, 6.5 to 19.0 times, 6.5 to 18.0 times,

[0653] 6.5 to 17.0 times, 6.5 to 16.0 times, 6.5 to 15.0 times, 6.5 to 14.0 times, 6.5 to 13.0 times, 6.5 to 12.0 times, 6.5 to 11.0 times, 6.5 to 10.0 times, 7.0 to 23.0 times, 7.0 to 22.0 times, 7.0 to 21.0 times, 7.0 to 20.0 times, 7.0 to 19.0 times, 7.0 to 18.0 times, 7.0 to 17.0 times, 7.0 to 16.0 times, 7.0 to 15.0 times, 7.0 to 14.0 times, 7.0 to 13.0 times, 7.0 to 12.0 times, 7.0 to 11.0 times, 7.0 to 10.0 times, 7.4 to 7.6 times, 7.3 to 7.7 times, 7.2 to 7.8 times, 7.1 to 7.9 times, 7.0 to 8.0 times, 6.9 to 8.1 times, 6.8 to 8.2 times, 6.7 to 8.3 times, 6.6 to 8.4 times, 6.5 to 8.5 times, 6.4 to 8.6 times, 6.3 to 8.7 times, 6.2 to 8.8 times, 6.1 to 8.9 times, 6.0 to 9.0 times, 5.9 to 9.1 times, 5.8 to 9.2 times, 5.7 to 9.3 times, 5.6 to 9.4 times, 5.5 to 9.5 times, 5.4 to 9.6 times, 5.3 to 9.7 times, 5.2 to 9.8 times, 5.1 to 9.9 times, 5.0 to 10.0 times, or some other multiple or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof, and the solvent can be an aprotic solvent, including but not limited to DMSO (including anhydrous DMSO).

[0654] Reductive amination

[0655] In some embodiments, activated saccharide and carrier protein are conjugated by reductive amination, which can be carried out by reacting the activated saccharide and carrier protein with a reducing agent.

[0656] In some embodiments, the reductive amination reaction is carried out in an aprotic solvent, such as DMSO which, in some embodiments, is anhydrous DMSO. In some embodiments, activated saccharide and carrier protein are compounded before addition of the reducing agent. As described above, such compounding can occur by lyophilizing activated saccharide and carrier protein, separately or together, from a lyophilization solution, followed by reconstituting the resulting lyophilizate with an aprotic solvent. If lyophilized and reconstituted together, reducing agent can thereafter be added to the reconstituted mixture of activated saccharide and carrier protein in aprotic solvent. If lyophilized and reconstituted separately in aprotic solvent, the reconstituted components can thereafter be combined and reducing agent added to the combined mixture. Similarly, if one or even both the components had been maintained separately in solution in aprotic solvent and not lyophilized, such solutions can be combined and reducing agent added to the combined mixture.

[0657] In some embodiments, the aprotic solvent in which the reductive amination reaction is carried out is dimethylsulphoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N- methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA), other aprotic solvents being possible. In some embodiments, the aprotic solvent is DMSO. In some embodiments, the aprotic solvent is anhydrous DMSO.

[0658] In some embodiments, the reducing agent used to carry out reductive amination of activated saccharide and carrier protein in aprotic solvent is sodium cyanoborohydride (NaCNBHs), sodium triacetoxyborohydride, sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, or an aminoborane, such as pyridine borane, 2-picoline-borane, 2,6- diborane-methanol, dimethylamine-borane, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), other reducing agents being possible. In some embodiments, the reducing agent can be sodium cyanoborohydride.

[0659] In some embodiments, reductive amination of compounded activated saccharide and carrier protein is carried out using any suitable mass of activated saccharide relative to mass of carrier protein, which, in some embodiments, is a mass ratio of at least or about 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3.5:1 , 3:1 , 2:1 , 1.9:1 , 1.8:1 , 1.7:1 , 1.6:1 , 1.5:1 , 1.4:1 , 1.3:1 , 1.2:1 , 1.1 :1 , 1.0:1 , 0.9:1 , 0.8:1 , 0.7:1 , 0.6:1 , 0.5:1 , 0.4:1 , 0.3:1 , 0.2:1 , 0.1 :1 , or some other mass ratio between or range encompassing any of the foregoing specifically enumerated mass ratios.

[0660] After reconstitution with aprotic solvent, the resolubilized activated saccharide and carrier protein can, in some embodiments, be preincubuted by combining and mixing the solutions in a reaction vessel for a period of time before addition of reducing agent. In some embodiments, the preincubation period can be at least or about 30, 45, 60, 75, 90, 105, or 120 minutes, or more, or an amount of time between or range encompassing any of the foregoing specifically enumerated periods of time, or some other period of time. In some embodiments, during the preincubation period, the mixture can be maintained at a suitable temperature which, in some embodiments, can be at least or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or 26°C, or a temperature between or range encompassing any of the foregoing specifically enumerated temperatures, or some other temperature. In some embodiments, after combining reconstituted activated saccharide and carrier protein, and whether or not a preincubation period occurred, reductive amination of activated saccharide and carrier protein can be initiated by addition to the combined solutions of an amount of reducing agent (e.g., NaCNBHs) which is sufficient to cause conjugation between the activated saccharide and carrier protein. In some embodiments, the amount of reducing agent (e.g., NaCNBHs) which is sufficient to cause conjugation between the activated saccharide and carrier protein is at least or about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1 , 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 molar equivalents of reducing agent to activated saccharide, or more, or some other number of molar equivalents between or range encompassing any of the foregoing specifically enumerated values.

[0661] In some embodiments, after addition of reducing agent (e.g., NaCNBHs), the reaction mixture can be incubated for a period of time sufficient for the conjugation reaction to go substantially to completion, or for some other desired period of time. In some embodiments, the period of incubation can be at least or about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours, or an amount of time between or range encompassing any of the foregoing specifically enumerated periods of time, or some other period of time. In some embodiments, during the incubation period, the mixture can be maintained at a suitable temperature which, in some embodiments, can be at least or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or 26°C, or a temperature between or range encompassing any of the foregoing specifically enumerated temperatures, or some other temperature.

[0662] In some embodiments, after the conjugation reaction has proceeded for sufficient time, the conjugation reaction can be terminated by addition to the mixture of an amount of a capping agent sufficient to reduce unreacted aldehydes in the activated saccharide back to hydroxyl groups. In some embodiments, the capping agent is sodium borohydride (NaBH4), other capping agents being possible. In some embodiments, the amount of capping agent (e.g., NaBH4) which is sufficient to terminate conjugation is at least or about 0.01 , 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.1 , 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 molar equivalents of capping agent to activated saccharide, or more, or some other number of molar equivalents between or range encompassing any of the foregoing specifically enumerated values.

[0663] In some embodiments, after addition of capping agent (e.g., NaBH4), the reaction mixture can be incubated for a period of time sufficient for the capping reaction to go substantially to completion, or for some other desired period of time. In some embodiments, the period of incubation can be at least or about 0.5, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours, or an amount of time between or range encompassing any of the foregoing specifically enumerated periods of time, or some other period of time. In some embodiments, during the incubation period, the mixture can be maintained at a suitable temperature which, in some embodiments, can be at least or about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or 26°C, or a temperature between or range encompassing any of the foregoing specifically enumerated temperatures, or some other temperature.

[0664] In some embodiments, the reductive amination reaction is carried out in a volume of aprotic solvent (e.g., DMSO or anhydrous DMSO) comprising suitable amounts of activated saccharide and carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), and a sufficient amount of reducing agent (e.g., NaCNBHs) to cause their conjugation by reductive amination. In some embodiments, the reductive amination reaction comprises at least or about 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 ,

[0665] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3,

[0666] 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5,

[0667] 7.6, 7.7, 7.8, 7.9, 8.0, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 grams of carrier protein (e.g.,

[0668] DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) per liter of solvent (e.g., DMSO or anhydrous DMSO), or more, or concentration between or range encompassing any of the foregoing specifically enumerated values, as well as comprising at least or about 1.0, 1.1 , 1.2,

[0669] 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4,

[0670] 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6,

[0671] 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,

[0672] 7.9, 8.0, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 grams of activated saccharide per liter of solvent (e.g., DMSO or anhydrous DMSO), or more, or concentration between or range encompassing any of the foregoing specifically enumerated values. In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,

[0673] 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2- 10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1 % w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1 .75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75- 1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0674] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide (e.g., from an S. pneumoniae serotype) and at least or about 1 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein. In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 1 g / L of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 1-20, 1-15, 1-14, 1-13, 1-12, 1-11 , 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 g / L carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0675] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2- 10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2- 20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 2 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 2-20, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4,

[0676] 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2- 10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,

[0677] 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0678] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3- 10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197)., or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 3 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 3-20, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, or 3-4 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,

[0679] 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25,

[0680] 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to serotypes 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11 A, 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0681] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4- 10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 4 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 4-20, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further com prise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0682] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 5 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 5-20, 5-15, 5-14, 5-13, 5-12, 5-11 , 5- 10, 5-9, 5-8, 5-7, or 5-6 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75-1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0683] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6- 10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 6 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 6-20, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, or 6-7 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In any of the foregoing embodiments, the reductive amination reaction can further comprise potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-

[0684] 7.5, or 2-5 mM potassium ions, and a trace amount of sodium ions, where trace amount is that amount which is attributable to typical contaminants in reagent grade or pharmaceutical grade chemical reagents, for example, less than 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, 0.0005 mM, 0.0001 mM, or lower concentration of sodium ions. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,

[0685] 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose. In any of the foregoing embodiments, the reductive amination reaction can further comprise at least 1 mM potassium ions, such as at least or about 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mM potassium ions, such as 2-20, 2-15, 2-10, 2-7.5, or 2-5 mM potassium ions, a trace amount of sodium ions, and sucrose, where the amount of sucrose is equal to or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% w / v, such as less than 5% w / v, less than 4% w / v, or less than 3% w / v. In any of the foregoing embodiments, the sufficient amount of reducing agent (e.g., NaCNBHs) can be at least or about 0.5 molar equivalents of the amount of activated saccharide, such as at least or about 0.5, 0.75, 1 , 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, or 3 molar equivalents, such as 0.5-3, 0.75-2, 0.75- 1.5, 0.75-1.25, or about 1 molar equivalents. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from any Streptococcus pneumoniae serotype, including but not limited to 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 any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 3. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 12F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 15A. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 23B. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 24F. In any of the foregoing embodiments, the activated saccharide can be prepared from bacterial capsular saccharides isolated from Streptococcus pneumoniae serotype 35B.

[0686] In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, and about, at least or greater than 1 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7- 10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 2 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 3 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 4 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 5 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 6 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7-9, or 7-8 g / L carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), or other amounts of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197). In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, at least or about 7 g / L of activated saccharide and at least or about 7 g / L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), such as, without limitation, about 7-20, 7-15, 7-14, 7-13, 7-12, 7-11 , 7-10, 7...

Claims

What is claimed is:

1. A method for producing a glycoconjugate by reductive amination, comprising reconstituting a lyophilized composition comprising a carrier protein and a potassium salt with DMSO, and reacting said reconstituted carrier protein with an activated bacterial capsular saccharide in DMSO in the presence of a reducing agent, wherein the concentration of soluble carrier protein in DMSO after reconstitution is at least 2 g / L.

2. The method of claim 1 , wherein said potassium salt is selected from the group consisting of potassium phosphate monobasic, potassium phosphate dibasic, and potassium chloride.

3. The method of any one of claims 1-2, wherein the molar ratio of potassium ions to carrier protein in said lyophilized composition is at least 30:1.

4. The method of any one of claims 1-3, wherein the molar ratio of potassium ions to carrier protein in said lyophilized composition ranges from about 30 to about 450.

5. The method of any one of claims 1-4, wherein said lyophilized composition comprises not more than an amount of sodium ions which is 90-fold greater on a molar basis than the amount of carrier protein in said lyophilized composition.

6. The method of any one of claims 1-5, wherein said lyophilized composition further comprises a non-reducing sugar.

7. The method of claim 6, wherein said non-reducing sugar is sucrose.

8. The method of claim 7, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is at least 2.5:1.

9. The method of claim 7, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is not more than 10:1.

10. The method of claim 7, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is not more than 7.5:1.

11. The method of any one of claims 1-10, wherein said lyophilized composition comprising carrier protein further comprises said activated bacterial capsular saccharide.

12. The method of any one of claims 1-10, further comprising, prior to said step of reacting, reconstituting a lyophilized composition comprising said activated bacterial capsular saccharide with DMSO and mixing said reconstituted composition comprising activated saccharide with said reconstituted composition comprising carrier protein.

13. The method of any one of claims 1-12, wherein said lyophilized composition comprising carrier protein and potassium salt is lyophilized from a lyophilization solution.

14. The method of claim 13, wherein said lyophilization solution comprises potassium phosphate buffer.

15. The method of claim 14, wherein the concentration of said potassium phosphate buffer is at least 2.5 mM.

16. The method of claim 15, wherein the concentration of said potassium phosphate buffer is at least 5 mM.

17. The method of claim 16, wherein the concentration of said potassium phosphate buffer is about 5 mM to about 90 mM.

18. The method of claim 17, wherein the concentration of said potassium phosphate buffer is about 5 mM to about 45 mM.

19. The method of claim 18, wherein the concentration of said potassium phosphate buffer is about 5 mM to about 15 mM.

20. The method of claim 19, wherein the concentration of said potassium phosphate buffer is about 5 mM to about 5 mM.

21. The method of any one of claims 14-20, wherein the pH of said potassium phosphate buffer ranges from about pH 7.0 to about pH 7.8.

22. The method of any one of claims 14-20, wherein the pH of said potassium phosphate buffer ranges from about pH 7.2 to about pH 7.6.

23. The method of any one of claims 14-20, wherein the pH of said potassium phosphate buffer is about pH 7.4.

24. The method of claim 13, wherein the concentration of potassium ions in said lyophilization solution is at least 2.5 mM.

25. The method of claim 24, wherein the concentration of potassium ions in said lyophilization solution is at least 5 mM.

26. The method of claim 25, wherein the concentration of potassium ions in said lyophilization solution is about 5 mM to about 90 mM.

27. The method of claim 26, wherein the concentration of potassium ions in said lyophilization solution is about 5 mM to about 45 mM.

28. The method of claim 27, wherein the concentration of potassium ions in said lyophilization solution is about 5 mM to about 15 mM.

29. The method of any one of claims 13-28, wherein said lyophilization solution further comprises sucrose.

30. The method of claim 29, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is at least 2.5:1.31 . The method of claim 29, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is not more than 10:1.

32. The method of claim 29, wherein the mass ratio of sucrose to carrier protein in said lyophilized composition is not more than 7.5:1.

33. The method of any one of claims 13-32, wherein said lyophilization solution comprises not more than 25 mM of sodium ions.

34. The method of any one of claims 13-32, wherein said lyophilization solution comprises not more than a trace amount of sodium ions.

35. The method of any one of claims 13-34, wherein said lyophilization solution comprises at least 3 g / L of carrier protein.

36. The method of any one of claims 13-35, wherein said lyophilization solution comprises 3- 12 g / L of carrier protein.

37. The method of any one of claims 13-36, wherein said lyophilization solution comprises 4- 11 g / L of carrier protein.

38. The method of any one of claims 13-37, wherein said lyophilization solution comprises 5- 10 g / L of carrier protein.

39. The method of any one of claims 13-38, wherein said lyophilization solution comprises 6- 9 g / L, 7-8 g / L, or about 7 g / L, or about 6 g / L of carrier protein.

40. The method of any one of claims 1-39, wherein said bacterial capsular saccharide is sized before activation.

41. The method of any one of claims 1-40, wherein said bacterial capsular saccharide is activated by reaction with an oxidizing agent.

42. The method of claim 41 , wherein said oxidizing agent is periodate.

43. The method of any one of claims 1-42, wherein the ratio of the amounts of activated bacterial capsular saccharide and carrier protein that are reacted ranges from about 3:1 to about 0.33:1.

44. The method of any one of claims 1-43, wherein the ratio of the amounts of activated bacterial capsular saccharide and carrier protein that are reacted ranges from about 2:1 to about 0.5:1.

45. The method of any one of claims 1-44, wherein the ratio of the amounts of activated bacterial capsular saccharide and carrier protein that are reacted ranges from about 1.5:1 to about 0.67:1.

46. The method of any one of claims 1-45, wherein the ratio of the amounts of activated bacterial capsular saccharide and carrier protein that are reacted is about 1:1.

47. The method of any one of claims 1-46, wherein said reductive amination reaction comprises at least 1 g / L of carrier protein and at least 1 g / L of activated bacterial capsular saccharide.

48. The method of any one of claims 1-47, wherein said reductive amination reaction comprises at least 2 g / L of carrier protein and at least 2 g / L of activated bacterial capsular saccharide.

49. The method of any one of claims 1-48, wherein said reductive amination reaction comprises at least 3 g / L of carrier protein and at least 3 g / L of activated bacterial capsular saccharide.

50. The method of any one of claims 1-49, wherein said reductive amination reaction comprises at least 4 g / L of carrier protein and at least 4 g / L of activated bacterial capsular saccharide.

51. The method of any one of claims 1-50, wherein said reductive amination reaction comprises at least 5 g / L of carrier protein and at least 5 g / L of activated bacterial capsular saccharide.

52. The method of any one of claims 1-51 , wherein said reducing agent is sodium cyanoborohydride.

53. The method of any one of claims 1-52, wherein said DMSO is anhydrous DMSO.

54. The method of any one of claims 1-53, further comprising purifying said glycoconjugate.

55. The method of claim 54, wherein said glycoconjugate is purified by ultrafiltration or diafiltration.

56. The method of any one of claims 1-55, wherein the yield of said glycoconjugate is at least 45%.

57. The method of any one of claims 1-56, wherein the yield of said glycoconjugate is at least 50%.

58. The method of any one of claims 1-57, wherein the yield of said glycoconjugate is at least 55%.

59. The method of any one of claims 1-58, wherein the yield of said glycoconjugate is at least 60%.

60. The method of any one of claims 1-59, wherein the yield of said glycoconjugate is at least 65%.61 . The method of any one of claims 1-60, wherein the yield of said glycoconjugate is at least 70%.

62. The method of any one of claims 1-61 , wherein the yield of said glycoconjugate is at least 75%.

63. The method of any one of claims 1-62, wherein said bacterial capsular saccharide is isolated from a Streptococcus pneumoniae bacteria of defined serotype.

64. The method of claim 63, wherein said serotype 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, 38, 72, and 73.

65. The method of any one of claims 1-55, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae bacteria serotype 3.

66. The method of claim 65, wherein said Streptococcus pneumoniae serotype 3 capsular saccharide is the acid form of said saccharide.

67. The method of claim 65, wherein said Streptococcus pneumoniae serotype 3 capsular saccharide is the sodium salt of said saccharide.

68. The method of claim 65, wherein said Streptococcus pneumoniae serotype 3 capsular saccharide is the potassium salt of said saccharide.

69. The method of any one of claims 65-68, wherein said reductive amination reaction comprises greater than 1 g / L of said activated Streptococcus pneumoniae serotype 3 capsular saccharide.

70. The method of any one of claims 65-69, wherein the yield of glycoconjugate, after purification, is at least 30%.

71. The method of any one of claims 1-55, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae bacteria serotype 12F.

72. The method of claim 71 , wherein said reductive amination reaction comprises greater than 1 g / L of said activated Streptococcus pneumoniae serotype 12F capsular saccharide.

73. The method of any one of claims 69-72, wherein the yield of glycoconjugate, after purification, is at least 50%.

74. The method of any one of claims 1-55, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae bacteria serotype 15A.

75. The method claim 74, wherein said reductive amination reaction comprises greater than 3 g / L of said activated Streptococcus pneumoniae serotype 15A capsular saccharide.

76. The method of any one of claims 72-75, wherein the yield of glycoconjugate, after purification, is at least 10%.

77. The method of any one of claims 1-55, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae bacteria serotype 23B.

78. The method claim 77, wherein said reductive amination reaction comprises greater than2 g / L of said activated Streptococcus pneumoniae serotype 23B capsular saccharide.

79. The method of any one of claims 75-78, wherein the yield of glycoconjugate, after purification, is at least 10%.

80. The method of any one of claims 1-55, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae bacteria serotype 35B.

81. The method claim 80, wherein said reductive amination reaction comprises greater than3 g / L of said activated Streptococcus pneumoniae serotype 35B capsular saccharide.

82. The method of any one of claims 78-81, wherein the yield of glycoconjugate, after purification, is at least 10%.

83. The method of any one of claims 1-82, wherein said carrier protein is selected from the group consisting of DT, TT, fHBP, PD, SCP, and CRM protein.

84. The method of any one of claims 1-83, wherein said CRM protein is CRM197.

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