[go: up one dir, main page]

WO2025219904A1 - Improved methods for producing glycoconjugates by reductive amination in aprotic solvent - Google Patents

Improved methods for producing glycoconjugates by reductive amination in aprotic solvent

Info

Publication number
WO2025219904A1
WO2025219904A1 PCT/IB2025/053995 IB2025053995W WO2025219904A1 WO 2025219904 A1 WO2025219904 A1 WO 2025219904A1 IB 2025053995 W IB2025053995 W IB 2025053995W WO 2025219904 A1 WO2025219904 A1 WO 2025219904A1
Authority
WO
WIPO (PCT)
Prior art keywords
carrier protein
kda
less
crm197
lyophilization
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/053995
Other languages
French (fr)
Inventor
Jianxin Gu
Justin Keith Moran
Suddham Singh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Corp Belgium, Pfizer Corp SRL filed Critical Pfizer Corp Belgium
Publication of WO2025219904A1 publication Critical patent/WO2025219904A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • 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.
  • 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.
  • E5. The method of E4, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria.
  • E6 The method of E4-E5, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria of defined serotype.
  • 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.
  • 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 k
  • 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,
  • 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.
  • 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
  • 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
  • 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,
  • 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.
  • mM millimolar
  • 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.
  • E32 The method of E18-E31 , wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).
  • 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
  • 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
  • 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.
  • 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.
  • E42 The method of E38-E41, wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).
  • 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.
  • mM millimolar
  • 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.
  • 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.
  • 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
  • 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%.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 16, 17, 18, 19, or 20 molar equivalents or ranges from about 0.01 to about 20 molar equivalents.
  • E74 The method of E73, wherein glycoconjugate is purified by ultrafiltration and/or diafiltration.
  • E83 An immunogenic composition comprising the glycoconjugate of E82.
  • 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.
  • 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.
  • 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.
  • saccharide refers to polysaccharide or oligosaccharide, and unless otherwise specified, may be used interchangeably.
  • 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.
  • 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.
  • 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).
  • bacterial capsular saccharide or polysaccharide
  • capsular saccharide or polysaccharide
  • capsular saccharide or polysaccharide
  • capsular saccharide or polysaccharide
  • 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.
  • 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.
  • 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.
  • 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.
  • the isolated capsular saccharide obtained by purification can be characterized by different parameters including, for example the weight average molecular weight (Mw).
  • Mw weight average molecular weight
  • the molecular weight of the polysaccharide can be measured by Size Exclusion Chromatography (SEC) combined with Multiangle Laser Light Scattering detector (MALLS).
  • 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.
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from a pathogenic Streptococcus, a pathogenic Neisseria or a pathogenic Escherichia.
  • 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 pulpe; Clostridium perfringens; Coccidioides immitis; Coccidioides posadasii; Cowdria ruminantium (Heartwater); Coxiella burnetii; Enterococcus faecalis;
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from Neisseria meningitidis, S. pneumoniae, S. agalactiae or Escherichia coli.
  • the capsular saccharide used in the present invention is a capsular saccharide from S. pneumoniae or S. agalactiae.
  • the capsular saccharide used in the present invention is a capsular saccharide from S. pneumoniae.
  • 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.
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from Neisseria meningitidis.
  • 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).
  • the capsular saccharide used in the present invention is a capsular saccharide from N.
  • 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).
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from Streptococcus agalactiae (Group B streptococcus (GBS)).
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from GBS types la, lb, II, III or V.
  • the capsular saccharide used in the present invention is a capsular saccharide from Escherichia coli.
  • 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).
  • ETEC Escherichia coli - enterotoxigenic
  • EPEC Escherichia coli - enteropathogenic
  • EHEC Escherichia coli - O157:H7 enterohemorrhagic
  • EIEC Escherichia coli - enteroinvasive
  • the capsular saccharide used in the present invention is
  • 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.
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O104:H4.
  • the capsular saccharide used in the present invention is a capsular saccharide from an Escherichia coli serotype O1 :K12:H7.
  • 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.
  • the capsular saccharide used in the present invention is a capsular saccharide from Steptococcus pneumoniae.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Glycoconjugates of the disclosure comprise a carrier protein to which saccharide is conjugated.
  • 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.
  • CRM cross-reactive material
  • 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 pi97 tox- 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 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.
  • the carrier protein of the glycoconjugate is Streptococcal C5a Peptidase (SCP).
  • 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.
  • GAS group A Streptococcus
  • GAS group B Streptococcus
  • the carrier protein of the glycoconjugate is an enzymatically inactive SCP from a GAS (SCPA), or fragment thereof.
  • SCPA GAS
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides methods of preparing glycoconjugates comprising saccharides conjugated to carrier protein.
  • the saccharides are bacterial capsular saccharides including, without limitation, bacterial capsular saccharides obtained from pathogenic strains of Streptococcus pneumoniae.
  • 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.
  • 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.
  • saccharides are sized to a target molecular weight or range before carrying out a conjugation reaction.
  • saccharides, whether or not sized are activated to increase their chemical reactivity with carrier protein.
  • saccharides are activated by reaction with an oxidizing agent, optionally followed by quenching the oxidation reaction by addition of a quenching agent.
  • activated saccharides and/or carrier protein can be lyophilized, separately or together.
  • 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.
  • sizing of certain saccharides to a target molecular weight range can be performed prior to the conjugation to a carrier protein.
  • 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.
  • the isolated capsular saccharide is not sized before its use in preparing a glycoconjugate as described herein.
  • 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.
  • chemical hydrolysis is conducted using diluted sulfuric acid. In some embodiments, chemical hydrolysis is conducted using diluted phosphoric acid. In some embodiments, chemical hydrolysis is conducted using diluted nitric acid. In some embodiments, chemical hydrolysis is conducted using diluted perchloric acid.
  • the size of the purified capsular saccharide can also be reduced by mechanical homogenization.
  • the size of the purified capsular saccharide is reduced by high pressure homogenization.
  • High pressure homogenization achieves high shear rates by pumping the process stream through a flow path with sufficiently small dimensions. The shear rate is increased by using a larger applied homogenization pressure, and exposure time can be increased by recirculating the feed stream through the homogenizer.
  • the high-pressure homogenization process can be appropriate for reducing the size of the purified capsular saccharide while preserving the structural features of the saccharide.
  • isolated capsular saccharide can be sized to any suitable weight average molecular weight prior to activation and conjugation.
  • 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 k
  • kDa
  • saccharides 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.
  • 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.
  • the oxidizing agent is any chemical, compound, or agent capable of oxidizing a terminal hydroxyl present in a saccharide to an aldehyde.
  • the oxidizing agent is periodate.
  • periodate refers to periodate and periodic acid, and includes both metaperiodate (IO4 ) and orthoperiodate (IOe 5 ) and the various salts of periodate (e.g., sodium periodate, potassium periodate, sodium metaperiodate, potassium metaperiodate, sodium orthoperiodate, and potassium orthoperiodate).
  • the oxidizing agent is periodate in the presence bivalent cations (e.g., Mg 2+ , Ca 2+ ).
  • bivalent cations e.g., Mg 2+ , Ca 2+ .
  • 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.
  • hydroxyl groups within the saccharide can be activated by TEMPO-mediated oxidation.
  • the saccharide which is activated in this way is bacterial capsular saccharide from S. pneumoniae serotype 12F, with others being possible.
  • 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.
  • the saccharide which is activated in this way is bacterial capsular saccharide from S. pneumoniae serotype 33F, with others being possible.
  • saccharide is reacted with an oxidizing agent, such as periodate, under conditions chosen to result in a desired degree of saccharide activation.
  • 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
  • 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.
  • oxidizing agent such as periodate
  • 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.
  • oxidizing agent such as periodate
  • 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.
  • oxidizing agent such as periodate
  • 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.
  • oxidizing agent such as periodate
  • the reaction with the oxidizing agent can be quenched by addition of a quenching agent to the reaction.
  • 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.
  • the quenching agent is selected from sodium and potassium salts of sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphite, or hypophosphite.
  • the quenching agent is a 1 ,2-aminoalcohol of formula (I): where R 1 is selected from H, methyl, ethyl, propyl or isopropyl.
  • 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.
  • such compounds have the formula (II): where R 1 and R 2 are each independently selected from H, methyl, ethyl, propyl or isopropyl.
  • the quenching agent is an amino acid including, for example, serine, threonine, cysteine, cystine, methionine, proline, hydroxyproline, tryptophan, tyrosine, and histidine.
  • 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.
  • 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.
  • the weight average molecular weight (Mw) of activated saccharide can be determined according to methods familiar to those of ordinary skill in the art.
  • 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
  • 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
  • carrier protein can also be activated chemically before carrying out 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.
  • 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.
  • both lyophilizates can be reconstituted in solvent and then compounded together before conjugation.
  • the combined lyophilizate can be reconstituted in solvent before proceeding to carry out the conjugation reaction.
  • 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.
  • activated saccharide and/or carrier protein can be maintained in a solution (lyophilization solution) which is subjected to lyophilization, producing a lyophilizate comprising either or both components.
  • 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.
  • lyophilization of certain carrier proteins such as CRM197
  • 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.
  • aprotic solvents such as dimethylsulfoxide (DMSO), e.g., below about 3 g/L
  • 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.
  • 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.
  • 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
  • the conjugation reaction is reductive amination.
  • lyophilization solution comprises one or more compounds which function as a pH buffer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises sucrose.
  • 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,
  • 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,
  • the potassium salt can be potassium phosphate monobasic (including hydrates thereof), and/or potassium phosphate dibasic (including hydrates thereof), and/or potassium chloride.
  • the potassium salt is potassium phosphate monobasic (including hydrates thereof), and/or potassium phosphate dibasic (including hydrates thereof), but no added potassium chloride.
  • 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.
  • 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,
  • 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.
  • the potassium salt can be potassium phosphate monobasic (including hydrates thereof), and/or potassium phosphate dibasic (including hydrates thereof), and/or potassium chloride.
  • 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.
  • 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
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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
  • 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
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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 + .
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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 ratio
  • the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197
  • 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
  • the lyophilization solution before lyophilization, 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.
  • 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,
  • the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.
  • 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.
  • the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 3-9 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, 5 mM to 40 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 3-9 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, 5 mM to 40 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 3-9 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, 5 mM to 40 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 3-9 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K + ions.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein, at least 5 mM K + ions and less than 25 mM Na + ions.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and 5 mM to 40 mM K + ions.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution comprises 5-7 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • the lyophilization solution before lyophilization, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the lyophilization solution before lyophilization, comprises about 6 g/L of CRM197 carrier protein and about 8.5 mM K + ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • lyophilizate formed following lyophilization of the lyophilization solution comprising carrier protein are described further below.
  • lyophilizate comprises potassium ions in specified molar ratio relative to the amount of carrier protein.
  • lyophilizate comprises potassium ions in specified molar ratio relative to an amount of sodium ions which are also present.
  • lyophilizate comprises sucrose in specified mass ratio relative to the amount of carrier protein.
  • the carrier protein can be a CRM protein, including but not limited to CRM197 protein and variants thereof.
  • 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
  • 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
  • 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,
  • lyophilizate comprising carrier protein may be reconstituted in aprotic solvent in preparation for conjugating carrier protein with activated saccharide, for example, by reductive amination.
  • the aprotic solvent comprises, consists essentially of, or consists of dimethylsulphoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, N-methyl-2-pyrrolidone, or hexamethylphosphoramide (HMPA).
  • the aprotic solvent comprises, consists essentially of, or consists of dimethylsulphoxide (DMSO) which, in some embodiments, can be anhydrous DMSO.
  • reconstituted lyophilizate comprises potassium ions in specified molar ratio relative to the amount of carrier protein.
  • 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.
  • reconstituted lyophilizate comprises potassium ions in specified molar ratio relative to an amount of sodium ions which are also present.
  • reconstituted lyophilizate comprises sucrose in specified mass ratio relative to the amount of carrier protein.
  • 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.
  • 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,
  • 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.
  • 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
  • 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).
  • 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, 4-10, 3-9, 4-25, 4-24, 4-23, 4-22, 4-21,
  • 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)
  • 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,
  • 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,
  • 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).
  • 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
  • the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, including but not limited to CRM197 and variants thereof
  • the solvent can be an aprotic solvent, including but not limited to DMSO (including anhydrous DMSO).
  • 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.
  • the reductive amination reaction is carried out in an aprotic solvent, such as DMSO which, in some embodiments, is anhydrous DMSO.
  • 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.
  • the reconstituted components can thereafter be combined and reducing agent added to the combined mixture.
  • reducing agent added to the combined mixture.
  • 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.
  • the aprotic solvent is DMSO.
  • the aprotic solvent is anhydrous DMSO.
  • 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.
  • the reducing agent can be sodium cyanoborohydride.
  • 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.
  • the resolubilized activated saccharide and carrier protein can, in some embodiments, be preincumped by combining and mixing the solutions in a reaction vessel for a period of time before addition of reducing agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • an amount of reducing agent e.g., NaCNBHs
  • 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.
  • reducing agent e.g.,
  • 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.
  • 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.
  • 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.
  • 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.
  • the capping agent is sodium borohydride (NaBH4), other capping agents being possible.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • suitable amounts of activated saccharide and carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • reducing agent e.g., NaCNBHs
  • 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 ,
  • DT, TT, fHBP, PD, SCP, or CRM protein such as CRM197
  • solvent e.g., DMSO or anhydrous DMSO
  • the reductive amination reaction can further comprise potassium ions.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions
  • activated saccharide e.g., from an S.
  • carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • CRM197 carrier protein
  • carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • 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).
  • aprotic solvent e.g.
  • 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.
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions
  • 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).
  • aprotic solvent e.g.
  • 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).
  • aprotic solvent e.g.
  • 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).
  • aprotic solvent e.g.
  • 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).
  • aprotic solvent e.g.
  • the reductive amination reaction can further comprise potassium ions.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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,
  • 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
  • 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.
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g.,
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., DT
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., DT
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., DT
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., DT
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., DT
  • the reductive amination reaction can further comprise potassium ions.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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,
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at
  • the reductive amination reaction can further com prise potassium ions.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, and
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 2
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 3
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 4
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 5
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 6
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 7
  • the reductive amination reaction can further comprise potassium ions.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, and about, at
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 2 g
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 3 g
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 4 g
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 5 g
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 6 g
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 7 g
  • the reductive amination reaction can further comprise potassium ions.
  • 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.
  • 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-
  • 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,
  • 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.
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, and about, at least or
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 2 g/L of
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 3 g/L of
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 4 g/L of
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 5 g/L of
  • 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 6 g/L of
  • 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, 7-9, 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).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions e.g., potassium ions, at least or about 7 g/L of
  • the reductive amination reaction can further comprise potassium ions.
  • 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.
  • 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.
  • 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,
  • 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,
  • 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.
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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-
  • 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.
  • 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.
  • 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.
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 1-2 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions 1-2 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • carrier protein e.g., DT, TT, fHBP,
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 2-3 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions 2-3 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • carrier protein e.g., DT, TT, fHBP, PD
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 3-4 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions 3-4 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • carrier protein e.g., DT, TT, fHBP,
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 4-5 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • potassium ions 4-5 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • carrier protein e.g., DT, TT, fHBP,
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 5-6 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • aprotic solvent e.g., DMSO or anhydrous DMSO
  • the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 6-7 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197).
  • the reductive amination reaction can further comprise potassium ions.
  • 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,
  • 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
  • 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.
  • the sufficient amount of reducing agent e.g., NaCNBHs
  • the sufficient amount of reducing agent 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.
  • 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.
  • 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.
  • 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.
  • bacterial capsular saccharide from Streptococcus pneumoniae serotype 3 is activated and conjugated with CRM197 protein by reductive amination.
  • activated Pn3 saccharide and CRM197 protein are lyophilized separately.
  • a CRM197 protein lyophilization solution is prepared containing of 6.0 ⁇ 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized.
  • the lyophilized activated Pn3 saccharide and CRM197 protein are each reconstituted with anhydrous DMSO.
  • the lyophilized activated Pn3 saccharide is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL.
  • the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL.
  • volumes of reconstituted activated Pn3 saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide and CRM197 protein each are respectively about 0.75 to 1.25 mg/mL, or about 1.0 mg/mL.
  • the combined solutions of reconstituted activated Pn3 saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ⁇ 2°C, during which time the moisture level of the mixture can be monitored.
  • the conjugation reaction is initiated by adding to the mixture 0.75 to 1.25 molar equivalents of the reducing agent NaCNBHs, or about 1.0 molar equivalents of NaCNBHs, relative to the amount of activated Pn3 saccharide in the reaction mixture.
  • the conjugation reaction is incubated for 24 ⁇ 4 hrs at 23 ⁇ 2°C.
  • the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH 4 .
  • the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ⁇ 2°C.
  • the solution containing the Pn3 saccharide-CRM ? protein conjugates is diluted and purified.
  • the activated Pn3 saccharide before conjugation, has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100- 150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range.
  • Mw average molecular weight
  • the activated Pn3 saccharide before conjugation, has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range.
  • DO degree of oxidation
  • the free saccharide content of PnS-CRM after conjugation, the free saccharide content of PnS-CRM ?
  • conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%.
  • the yield of PnS-CRM ? conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the average molecular weight (Mw) of Pn3-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range.
  • the activated Pn3 saccharide before conjugation, has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 10-18 or about 12-16 or about 13-15. In some embodiments, after conjugation, the free saccharide content of Pn3-CRM 7 conjugates is less than 10% and the yield is at least 70%. In some embodiments, after conjugation, the average Mw of Pn3-CRM 7 conjugates is about 1500-3500 kDa or about 1750-3250 kDa or about 3000-3500 kDa.
  • bacterial capsular saccharide from Streptococcus pneumoniae serotype 12F is activated and conjugated with CRM197 protein by reductive amination.
  • activated Pn12F saccharide and CRM197 protein are lyophilized separately.
  • a CRM197 protein lyophilization solution is prepared containing of 6.0 ⁇ 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized.
  • the lyophilized activated Pn12F saccharide and CRM197 protein are each reconstituted with anhydrous DMSO.
  • the lyophilized activated Pn12F saccharide is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL.
  • the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 3.0 mg/mL, or about 2.5 mg/mL.
  • volume of reconstituted activated Pn12F saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 0.75 to 1.25 mg/mL, or about 1.0 mg/mL, and the final concentration of CRM197 protein is about 0.75 to 1.5 mg/mL, or about 1.25 mg/mL.
  • the combined solutions of reconstituted activated Pn12F saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ⁇ 2°C, during which time the moisture level of the mixture can be monitored.
  • the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1.0 molar equivalents of NaCNBHs, relative to the amount of activated Pn12F saccharide in the reaction mixture.
  • the conjugation reaction is incubated for 24 ⁇ 4 hrs at 23 ⁇ 2°C.
  • the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBFL, or about 2.0 molar equivalents of NaBFL.
  • the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ⁇ 2°C.
  • the solution containing the Pn12F saccharide-CRM ? protein conjugates is diluted and purified.
  • the activated Pn12F saccharide before conjugation, has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range.
  • Mw average molecular weight
  • the activated Pn12F saccharide before conjugation, has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11, 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16- 18, 17-18, or some other degree of oxidation or range.
  • DO degree of oxidation
  • the free saccharide content of Pn12F-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%.
  • the yield of Pn12F-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the average molecular weight (Mw) of Pn12F- CRM197 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250- 1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250- 3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range.
  • the activated Pn12F saccharide before conjugation, has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 3-8 or about 4-7 or about 5-6. In some embodiments, after conjugation, the free saccharide content of Pn12F-CRM 7 conjugates is less than 10% and the yield is at least 70%. In some embodiments, after conjugation, the average Mw of Pn12F- CRM197 conjugates is about 2500-4500 kDa or about 3000-4250 kDa.
  • bacterial capsular saccharide from Streptococcus pneumoniae serotype 15A is activated and conjugated with CRM197 protein by reductive amination.
  • activated Pn15A saccharide and CRM197 protein are lyophilized separately.
  • a CRM197 protein lyophilization solution is prepared containing of 6.0 ⁇ 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized.
  • the lyophilized activated Pn15A saccharide and CRM197 protein are each reconstituted with anhydrous DMSO.
  • the lyophilized activated Pn15A saccharide is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL.
  • the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL.
  • volume of reconstituted activated Pn15A saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL, and the final concentration of CRM197 protein is about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL.
  • the combined solutions of reconstituted activated Pn15A saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ⁇ 2°C, during which time the moisture level of the mixture can be monitored.
  • the conjugation reaction is initiated by adding to the mixture 0.75 to 1.25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn15A saccharide in the reaction mixture.
  • the conjugation reaction is incubated for 24 ⁇ 4 hrs at 23 ⁇ 2°C.
  • the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH4.
  • the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ⁇ 2°C.
  • the solution containing the Pn15A saccharide-CRM ? protein conjugates is diluted and purified.
  • the activated Pn15A saccharide before conjugation, has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range.
  • Mw average molecular weight
  • the activated Pn15A saccharide before conjugation, has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16- 18, 17-18, or some other degree of oxidation or range.
  • DO degree of oxidation
  • the free saccharide content of Pn15A-CRM 7 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%.
  • the yield of Pn15A-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the average molecular weight (Mw) of Pn15A- CRM197 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250- 1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250- 3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range.
  • the activated Pn15A saccharide before conjugation, has Mw of about 75-225 kDa or about 100-200 kDa and DO of about 3-7 or about 4-7 or about 4-6. In some embodiments, after conjugation, the free saccharide content of Pn15A-CRM 7 conjugates is less than 10% and the yield is at least 60%. In some embodiments, after conjugation, the average Mw of Pn15A- CRM197 conjugates is about 1250-2750 kDa or about 1500-2500 kDa or about 1750-2250 kDa.
  • bacterial capsular saccharide from Streptococcus pneumoniae serotype 23B is activated and conjugated with CRM197 protein by reductive amination.
  • activated Pn23B saccharide and CRM197 protein are lyophilized separately.
  • a CRM197 protein lyophilization solution is prepared containing of 6.0 ⁇ 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized.
  • the lyophilized activated Pn23B saccharide and CRM197 protein are each reconstituted with anhydrous DMSO.
  • the lyophilized activated Pn23B saccharide is reconstituted with anhydrous DMSO to a concentration of about 5.0 to 7.0 mg/mL, or about 6.0 mg/mL.
  • the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 5.0 to 8.0 mg/mL, or about 6.6 mg/mL.
  • volume of reconstituted activated Pn23B saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 2.5 to 3.5 mg/mL, or about 3.0 mg/mL, and the final concentration of CRM197 protein is about 2.5 to 4.0, or about 3.3 mg/mL.
  • the combined solutions of reconstituted activated Pn23B saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ⁇ 2°C, during which time the moisture level of the mixture can be monitored.
  • the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn23B saccharide in the reaction mixture.
  • the conjugation reaction is incubated for 24 ⁇ 4 hrs at 23 ⁇ 2°C.
  • the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH 4 .
  • the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ⁇ 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn23B saccharide-CRMi97 protein conjugates is diluted and purified.
  • the activated Pn23B saccharide before conjugation, has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20- 50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200- 300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range.
  • Mw average molecular weight
  • the activated Pn23B saccharide before conjugation, has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range.
  • DO degree of oxidation
  • the free saccharide content of Pn23B-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%.
  • the yield of Pn23B-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the average molecular weight (Mw) of Pn23B-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500- 2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500- 4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range.
  • the activated Pn23B saccharide before conjugation, has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 7-11 or about 8-10. In some embodiments, after conjugation, the free saccharide content of Pn23B-CRMi97 conjugates is less than 10% and the yield is at least 50%. In some embodiments, after conjugation, the average Mw of Pn23B-CRMi97 conjugates is about 750-1500 or about 750-1250 kDa.
  • bacterial capsular saccharide from Streptococcus pneumoniae serotype 35B is activated and conjugated with CRM197 protein by reductive amination.
  • activated Pn35B saccharide and CRM197 protein are lyophilized separately.
  • a CRM197 protein lyophilization solution is prepared containing of 6.0 ⁇ 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized.
  • the lyophilized activated Pn35B saccharide and CRM197 protein are each reconstituted with anhydrous DMSO.
  • the lyophilized activated Pn35B saccharide is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL.
  • the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 8.0 to 12.0 mg/mL, or about 10.0 mg/mL.
  • volume of reconstituted activated Pn35B saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL, and the final concentration of CRM197 protein is about 4.0 to 6.0, or about 5.0 mg/mL.
  • the combined solutions of reconstituted activated Pn35B saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ⁇ 2°C, during which time the moisture level of the mixture can be monitored.
  • the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn35B saccharide in the reaction mixture.
  • the conjugation reaction is incubated for 24 ⁇ 4 hrs at 23 ⁇ 2°C.
  • the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH 4 .
  • the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ⁇ 2°C.
  • the solution containing the Pn35B saccharide-CRMi97 protein conjugates is diluted and purified.
  • the activated Pn35B saccharide before conjugation, has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20- 50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200- 300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range.
  • Mw average molecular weight
  • the activated Pn35B saccharide before conjugation, has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range.
  • DO degree of oxidation
  • the free saccharide content of Pn35B-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%.
  • the yield of Pn35B-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.
  • the average molecular weight (Mw) of Pn35B-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500- 2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500- 4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range.
  • the activated Pn35B saccharide before conjugation, has Mw of about 20-80 kDa or about 30-60 kDa or about 30-50 kDa and DO of about 6-10 or about 7-9. In some embodiments, after conjugation, the free saccharide content of Pn35B-CRMi97 conjugates is less than 10% and the yield is at least 55%. In some embodiments, after conjugation, the average Mw of Pn35B-CRMi97 conjugates is about 750-2500 or about 1000-2250 or about 1000-2000 kDa.
  • glycoconjugates can be purified, partially or fully, by a variety of techniques known to the skilled person. These techniques include, without limitation, dialysis, concentration/diafiltration operations against various buffers, tangential flow filtration precipitation/elution, column chromatography (DEAE or hydrophobic interaction chromatography, others being possible), membrane filtration, and depth filtration. Accordingly, in some embodiments, methods of the disclosure for producing a capsular saccharide glycoconjugate further comprise the step of purifying the glycoconjugate, partially or fully, after it is produced.
  • a reaction mixture containing conjugates of saccharide and carrier protein can be diluted with a larger volume of a dilution solution.
  • the dilution solution can be a chilled 5 mM succinate-saline solution at pH 6.0, and the conjugation reaction mixture can be diluted with a 4x-9x volume of such dilution solution.
  • the diluted conjugation reaction mixture can be mixed for a period of time, such as 10 minutes, or some other time period, and then subjected to microfiltration, such as filtration through a 5 pm filter, or some other filter.
  • the diluted conjugation reaction mixture can be concentrated by diafiltration.
  • diafiltration can be performed against the same solution as the dilution solution.
  • the diafiltration solution can be a 5 mM succinate-saline solution at pH 6.0.
  • diafiltration is performed using a membrane filter with a certain molecular weight cutoff (MWCO), such as 100 kDa MWCO, or some other MWCO.
  • MWCO molecular weight cutoff
  • the membrane filter is a regenerated cellulose membrane filter.
  • diafiltration is performed under conditions where the membrane challenge is 1-10 grams saccharide per ft 2 membrane area, transmembrane pressure is 5-20 PSI, and the retentate concentration is 1-5 g/L.
  • the concentrated conjugate solution is subjected to microfiltration, such as filtration through a 0.45 pm filter followed by filtration through a 0.22 pm filter, or some other filter.
  • the weight average molecular weight (Mw) of glycoconjugates prepared by methods of the disclosure can be determined according to methods familiar to those of ordinary skill in the art.
  • the weight average molecular weight (Mw) of glycoconjugates prepared by methods of the disclosure is at least or about 250 kilodaltons (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, 1050 kDa
  • the weight average molecular weight (Mw) of glycoconjugates prepared by methods of the disclosure can range from about 250-20000 kDa, 250-15000 kDa, 250-10000 kDa, 250-9000 kDa, 250-8000 kDa, 250-7000 kDa, 250-6500 kDa, 250-6000 kDa, 250-5500 kDa, 250-5400 kDa, 250-5300 kDa, 250-5200 kDa, 250-5100 kDa, 250-5000 kDa, 250- 4900 kDa, 250-4800 kDa, 250-4700 kDa, 250-4600 kDa, 250-4500 kDa, 250-4400 kDa, 250-
  • glycoconjugates of the disclosure can be characterized by the degree of conjugation of the carrier protein, which is the average number of lysine residues in the carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) that are conjugated to saccharide in a sample or preparation of glycoconjugate.
  • Degree of conjugation of carrier protein of a glycoconjugate can be determined by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate materials.
  • the degree of conjugation of carrier protein in glycoconjugates 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.
  • the degree of conjugation of carrier protein in glycoconjugates can be about 2-20,
  • glycoconjugates of the disclosure can be characterized by the ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of a glycoconjugate on a weight to weight (w/w) basis.
  • the ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of a glycoconjugate can be determined using routine methods known to those of skill in the art.
  • the ratio of saccharide to carrier protein e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • the ratio of saccharide to carrier protein is at least or about 0.5, 0.6, 0.7, 0.8, 0.9, 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,
  • the ratio of saccharide to carrier protein e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • the ratio of saccharide to carrier protein in a sample or preparation of glycoconjugate on a weight by weight basis can be about 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.5-1.0, 0.5-1.1 , 0.5-1.2, 0.5-1.3, 0.5-1.4, 0.5-1.5, 0.5-1.6, 0.5-1.7, 0.5-1.8, 0.5-1.9, 0.5-2.0, 0.5-2.1 , 0.5-2.2, 0.5-2.3, 0.5-2.4, 0.5-
  • glycoconjugates of the disclosure can be characterized by the amount of saccharide in a sample or preparation of a glycoconjugate which is free on a percent basis.
  • the amount of saccharide in a sample or preparation of a glycoconjugate which is free can be determined using routine methods known to those of skill in the art.
  • the percentage of saccharide in a sample or preparation of a glycoconjugate which is free saccharide is less than or about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.
  • glycoconjugates of the disclosure can be characterized by the number of covalent linkages between the carrier protein and the saccharide as a function of the number of repeat units of the saccharide.
  • a saccharide repeat unit may comprise one or more monosaccharides of same or different type which are covalently linked in a consistent way to form a structural unit which in turn is covalently linked in a consistent way to others of the same type repeatedly to form the saccharide polymer.
  • Saccharide repeat units may also comprise chemical groups other than monosaccharides, for examples, phosphate groups, other groups being possible.
  • Capsular saccharides can be distinguished both by differences among the sizes, constituents, and structures of their respective repeat units, and/or in the way in which repeat units are linked to each other forming the saccharide.
  • glycoconjugates can comprise at least 1 covalent linkage between the carrier protein and the saccharide per every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or more saccharide repeat units of the saccharide, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.
  • the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197.
  • glycoconjugates of the disclosure can be characterized by their molecular size distribution (Kd), which can be determined in a variety of ways familiar to those of ordinary skill in the art.
  • Kd molecular size distribution
  • size exclusion chromatography media e.g., CL-4B
  • Size exclusion chromatography (SEC) can be used in gravity fed columns to profile the molecular size distribution of conjugates. Large molecules excluded from the pores in the media elute more quickly than small molecules.
  • Fraction collectors can be used to collect the column eluate. The fractions can be tested colorimetrically by saccharide assay or by other methods.
  • At least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of glycoconjugates have a Kd below or equal to 0.3, for example, as determined using a CL-4B column.
  • the yield of glycoconjugate produced by carrying out a conjugation reaction using activated saccharide and carrier protein as described herein can be determined and expressed as percentage yield.
  • the yield of glycoconjugate can be determined using routine methods known to those of skill in the art.
  • the yield of glycoconjugate produced by carrying out a conjugation reaction using activated saccharide and carrier protein as described herein is at least or about 45%, 46%, 47%, 48%,
  • the disclosure provides an immunogenic composition comprising at least one glycoconjugate produced according to the methods described herein.
  • immunogenic compositions can be used in the prevention and/or treatment of diseases or disorders in a subject caused by microorganisms, such as bacteria, which produce the same or similar type of saccharide which is present in the glycoconjugate in the immunogenic composition.
  • the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates of at least two distinct types, the first comprising a first saccharide and the second comprising a second saccharide which is immunologically distinct from the first saccharide.
  • the carrier protein of the first and at least second glycoconjugates is the same type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the carrier protein of the first and at least second glycoconjugates are different types of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
  • the saccharides of the first and at least second glycoconjugates are from distinct serotypes of the same species of bacteria, whereas in other embodiments, the saccharides of the first and at least second glycoconjugates are from distinct species of bacteria. In some embodiments, the saccharides of the first and at least second glycoconjugates are from distinct serotypes of S. pneumoniae bacteria.
  • the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 20 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 20 or more serotypes of S.
  • pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible.
  • the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
  • a single type of carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
  • the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 21 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 21 or more serotypes of S.
  • pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible.
  • the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
  • a single type of carrier protein e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197
  • the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

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

IMPROVED METHODS FOR PRODUCING GLYCOCONJUGATES BY REDUCTIVE AMINATION IN APROTIC SOLVENT
BACKGROUND
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.
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.
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.
SUMMARY
Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following embodiments (E).
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.
E2. The method of E1 , wherein said carrier protein is selected from the group consisting of
DT, TT, fHBP, PD, SCP, and CRM protein. E3. The method of E2, wherein said CRM protein is CRM197.
E4. The method of any of the preceding embodiments, wherein said saccharide is a bacterial capsular saccharide.
E5. The method of E4, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria.
E6. The method of E4-E5, wherein said bacterial capsular saccharide is isolated from Streptococcus pneumoniae (Spn) bacteria of defined serotype.
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.
E8. The method of any of the preceding embodiments, wherein said saccharide is sized.
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.
E10. The method of any of the preceding embodiments, wherein said saccharide is activated by reaction with an oxidizing agent.
E11. The method of E10, wherein said oxidizing agent is periodate.
E12. The method of E11 , wherein said periodate is metaperiodate, or salts thereof.
E13. The method of E11 , wherein said periodate is orthoperiodate, or salts thereof.
E14. The method of E10-E13, wherein after reaction with oxidizing agent, said saccharide is contacted with a quenching agent.
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.
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.
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.
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.
E19. The method of E17-E18, wherein said activated saccharide or carrier protein is reconstituted with an aprotic solvent.
E20. The method of E19, wherein said aprotic solvent is dimethylsulfoxide (DMSO).
E21. The method of E20, wherein said DMSO is anhydrous.
E22. The method of E18-E21, wherein said lyophilized composition comprising carrier protein further comprises a potassium salt.
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.
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.
E25. The method of E22-E24, wherein said lyophilized composition comprising carrier protein comprises no more than trace amounts of sodium.
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.
E28. The method of E27, wherein said non-reducing sugar is sucrose.
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,
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.
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.
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.
E32. The method of E18-E31 , wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).
E33. The method of E18-E32, wherein after reconstitution the formerly lyophilized composition comprising carrier protein comprises not more than 25 mM sodium ions.
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.
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 .
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.
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,
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.
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.
E39. The method of E38, wherein said lyophilization solution is an aqueous solution further comprising potassium phosphate buffer.
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.
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.
E42. The method of E38-E41, wherein said carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein (such as CRM197).
E43. The method of E38-E42, wherein said CRM protein is CRM197.
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.
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.
E47. The method of E38-E46, wherein said lyophilization solution comprises not more than a trace amount of sodium ions.
E48. The method of E38-E47, wherein said lyophilization solution comprises about 5-40 mM potassium ions.
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.
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.
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.
E52. The method of E38-E51, wherein said lyophilization solution further comprises a nonreducing sugar.
E53. The method of E52, wherein said non-reducing sugar is sucrose.
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%.
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.
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.
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.
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.
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.
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.
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.
E62. The method of any of the preceding embodiments, wherein said reducing agent is sodium cyanoborohydride.
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.
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.
E65. The method of E64, further comprising adding said reducing agent to said mixture.
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.
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.
E68. The method of E67, wherein said capping agent is sodium borohydride.
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,
16, 17, 18, 19, or 20 molar equivalents, or ranges from about 0.01 to about 20 molar equivalents.
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 ,
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,
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,
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,
17, 18, 19, or 20 g/L, or ranges from about 1.0 g/L to about 20 g/L.
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,
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,
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,
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,
16, 17, 18, 19, or 20 g/L, or ranges from about 1.0 g/L to about 20 g/L.
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.
E73. The method of any of the preceding embodiments, further comprising purifying the glycoconjugate.
E74. The method of E73, wherein glycoconjugate is purified by ultrafiltration and/or diafiltration.
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.
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.
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%.
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.
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.
E81. The method of any of the preceding embodiments, wherein the yield, after purification of said glycoconjugate, is at least 45%.
E82. A glycoconjugate prepared by the method of any one of embodiments E1-E81.
E83. An immunogenic composition comprising the glycoconjugate of E82.
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.
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.
DETAILED DESCRIPTION
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.
Saccharides
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.
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.
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.
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.
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.
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.
The isolated capsular saccharide obtained by purification can be characterized by different parameters including, for example the weight average molecular weight (Mw). The molecular weight of the polysaccharide can be measured by Size Exclusion Chromatography (SEC) combined with Multiangle Laser Light Scattering detector (MALLS).
In some embodiments, the capsular saccharide used in the method of making or part of 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.
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.
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.
In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Staphylococcus aureus. In some embodiments, the capsular saccharide used in the present invention is a capsular saccharide from Staphylococcus aureus type 5 or Staphylococcus aureus type 8.
In some embodiments, the capsular saccharide used in the present invention is is a capsular saccharide from Enterococcus faecalis. In yet a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from is Haemophilus influenzae type b.
In a further embodiment, the capsular saccharide used in the present invention is a 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).
In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Escherichia coli. In a further embodiment, the capsular saccharide used in the present invention is a capsular saccharide from Enterococcus faecalis.
In a further embodiment, the capsular saccharide used in the present invention is a 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.
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).
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.
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.
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.
Carrier Protein
Glycoconjugates of the disclosure comprise a carrier protein to which saccharide is conjugated.
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.
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.
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).
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.
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.
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.
Methods of preparing glycoconjugates
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.
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.
Saccharide sizing
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.
In some embodiments, the size of the purified capsular saccharide is reduced by chemical hydrolysis. Chemical hydrolysis maybe conducted using a mild acid (e.g., acetic acid, formic acid, propanoic acid). In some embodiments, chemical hydrolysis is conducted using formic acid. In an embodiement, chemical hydrolysis is conducted using propanoic acid. In some embodiments, chemical hydrolysis is conducted using acetic acid. Chemical hydrolysis may also be conducted using a diluted strong acid (such as diluted hydrochloric acid, diluted sulfuric acid, diluted phosphoric acid, diluted nitric acid, or diluted perchloric acid). In some embodiments, chemical hydrolysis is conducted using diluted hydrochloric acid. In some embodiments, chemical hydrolysis is conducted using diluted sulfuric acid. In some embodiments, chemical hydrolysis is conducted using diluted phosphoric acid. In some embodiments, chemical hydrolysis is conducted using diluted nitric acid. In some embodiments, chemical hydrolysis is conducted using diluted perchloric acid.
The size of the purified capsular saccharide can also be reduced by mechanical homogenization. In some embodiments, the size of the purified capsular saccharide is reduced by high pressure homogenization. High pressure homogenization achieves high shear rates by pumping the process stream through a flow path with sufficiently small dimensions. The shear rate is increased by using a larger applied homogenization pressure, and exposure time can be increased by recirculating the feed stream through the homogenizer.
The high-pressure homogenization process can be appropriate for reducing the size of the purified capsular saccharide while preserving the structural features of the saccharide.
In some embodiments, 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.
Saccharide activation
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.
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.
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.
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.
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.
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.
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.
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.
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 R1 is 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 R1 and R2 are 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.
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.
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.
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.
Lyophilization
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.
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.
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.
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.
Lyophilization solution
Various non-limiting embodiments of lyophilization solution are described further below. pH
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,
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,
8.5, 9, 9.5, or 10, or value between or range encompassing any of the foregoing specifically enumerated pH values as end points.
Potassium ion concentration
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,
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,
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,
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,
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,
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,
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.
Potassium and sodium ion concentration
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,
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,
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,
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,
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,
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,
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.
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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
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+.
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+.
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
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+.
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+.
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+.
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+.
Molar ratio of potassium ions to carrier protein
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
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.
Molar ratio of potassium ions to sodium ions
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,
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.
Sucrose concentration
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.
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,
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,
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,
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,
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,
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.
Carrier protein concentration
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.
Certain additional non-limiting embodiments
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 at least 2.5 times the amount of CRM197 protein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K+ ions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 at least 2.5 times the amount of CRM197 protein.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 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.
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 at least 2.5 times the amount of CRM197 protein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K+ 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 less than 25 mM Na+ ions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K+ ions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 less than 25 mM Na+ ions.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 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.
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 2.5 to 10 times the amount of CRM197 protein.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 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, 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K+ ions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 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, 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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and at least 5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 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, 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises about 6 g/L of CRM197 carrier protein and at least 5 mM K+ ions.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 3-9 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 less than 25 mM Na+ ions.
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.
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 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, 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.
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.
In some embodiments, before lyophilization, the lyophilization solution comprises 4-8 g/L of CRM197 carrier protein and about 8.5 mM K+ 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 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.
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 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, 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 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Lyophilizate
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.
Molar ratio of potassium ions to carrier protein
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.
Molar ratio of potassium ions to sodium ions
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.
Sucrose concentration
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,
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 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,
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,
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,
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,
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.
Reconstituted lyophilizate
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.
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,
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,
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,
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,
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,
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,
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.
Molar ratio of potassium ions to carrier protein
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
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 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
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 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).
Carrier protein solubility
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,
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,
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,
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,
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,
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,
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.
Molar ratio of potassium ions to sodium ions
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).
Sucrose concentration
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,
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,
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,
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).
Reductive amination
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 ,
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,
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, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 grams of carrier protein (e.g.,
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,
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,
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,
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, 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,
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.
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.
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,
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.
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,
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, 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.
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.
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.
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-
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.
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, 7-9, 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 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.
In some embodiments, the reductive amination reaction comprises aprotic solvent (e.g., DMSO or anhydrous DMSO), potassium ions, 1-2 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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, 2-3 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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, 3-4 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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, 4-5 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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, 5-6 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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, 6-7 g/L of activated saccharide and 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11 , or 11-12 g/L 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.
In some embodiments, bacterial capsular saccharide from Streptococcus pneumoniae serotype 3 (Pn3) is activated and conjugated with CRM197 protein by reductive amination. In some embodiments, before conjugation, activated Pn3 saccharide and CRM197 protein are lyophilized separately. In some embodiments, before lyophilization, a CRM197 protein lyophilization solution is prepared containing of 6.0 ± 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized. In some embodiments, before conjugation, the lyophilized activated Pn3 saccharide and CRM197 protein are each reconstituted with anhydrous DMSO. In some embodiments, the lyophilized activated Pn3 saccharide is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL. In some embodiments, the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL. In some embodiments, after reconstitution, volumes of reconstituted activated Pn3 saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide and CRM197 protein each are respectively about 0.75 to 1.25 mg/mL, or about 1.0 mg/mL. In some embodiments, the combined solutions of reconstituted activated Pn3 saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ± 2°C, during which time the moisture level of the mixture can be monitored. In some embodiments, after the preincubation period, the conjugation reaction is initiated by adding to the mixture 0.75 to 1.25 molar equivalents of the reducing agent NaCNBHs, or about 1.0 molar equivalents of NaCNBHs, relative to the amount of activated Pn3 saccharide in the reaction mixture. In some embodiments, after the addition of the reducing agent, the conjugation reaction is incubated for 24 ± 4 hrs at 23 ± 2°C. In some embodiments, after the conjugation reaction has proceeded for the desired amount of time, the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH4. In some embodiments, after the addition of the capping agent, the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ± 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn3 saccharide-CRM ? protein conjugates is diluted and purified. In some embodiments, before conjugation, the activated Pn3 saccharide has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100- 150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn3 saccharide has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range. In some embodiments, after conjugation, the free saccharide content of PnS-CRM ? conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. In some embodiments, after conjugation, the yield of PnS-CRM ? conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, after conjugation, the average molecular weight (Mw) of Pn3-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn3 saccharide has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 10-18 or about 12-16 or about 13-15. In some embodiments, after conjugation, the free saccharide content of Pn3-CRM 7 conjugates is less than 10% and the yield is at least 70%. In some embodiments, after conjugation, the average Mw of Pn3-CRM 7 conjugates is about 1500-3500 kDa or about 1750-3250 kDa or about 3000-3500 kDa.
In some embodiments, bacterial capsular saccharide from Streptococcus pneumoniae serotype 12F (Pn12F) is activated and conjugated with CRM197 protein by reductive amination. In some embodiments, before conjugation, activated Pn12F saccharide and CRM197 protein are lyophilized separately. In some embodiments, before lyophilization, a CRM197 protein lyophilization solution is prepared containing of 6.0 ± 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized. In some embodiments, before conjugation, the lyophilized activated Pn12F saccharide and CRM197 protein are each reconstituted with anhydrous DMSO. In some embodiments, the lyophilized activated Pn12F saccharide is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 2.5 mg/mL, or about 2.0 mg/mL. In some embodiments, the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 1.5 to 3.0 mg/mL, or about 2.5 mg/mL. In some embodiments, after reconstitution, volumes of reconstituted activated Pn12F saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 0.75 to 1.25 mg/mL, or about 1.0 mg/mL, and the final concentration of CRM197 protein is about 0.75 to 1.5 mg/mL, or about 1.25 mg/mL. In some embodiments, the combined solutions of reconstituted activated Pn12F saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ± 2°C, during which time the moisture level of the mixture can be monitored. In some embodiments, after the preincubation period, the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1.0 molar equivalents of NaCNBHs, relative to the amount of activated Pn12F saccharide in the reaction mixture. In some embodiments, after the addition of the reducing agent, the conjugation reaction is incubated for 24 ± 4 hrs at 23 ± 2°C. In some embodiments, after the conjugation reaction has proceeded for the desired amount of time, the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBFL, or about 2.0 molar equivalents of NaBFL. In some embodiments, after the addition of the capping agent, the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ± 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn12F saccharide-CRM ? protein conjugates is diluted and purified. In some embodiments, before conjugation, the activated Pn12F saccharide has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn12F saccharide has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11, 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16- 18, 17-18, or some other degree of oxidation or range. In some embodiments, after conjugation, the free saccharide content of Pn12F-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. In some embodiments, after conjugation, the yield of Pn12F-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, after conjugation, the average molecular weight (Mw) of Pn12F- CRM197 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250- 1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250- 3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn12F saccharide has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 3-8 or about 4-7 or about 5-6. In some embodiments, after conjugation, the free saccharide content of Pn12F-CRM 7 conjugates is less than 10% and the yield is at least 70%. In some embodiments, after conjugation, the average Mw of Pn12F- CRM197 conjugates is about 2500-4500 kDa or about 3000-4250 kDa.
In some embodiments, bacterial capsular saccharide from Streptococcus pneumoniae serotype 15A (Pn15A) is activated and conjugated with CRM197 protein by reductive amination. In some embodiments, before conjugation, activated Pn15A saccharide and CRM197 protein are lyophilized separately. In some embodiments, before lyophilization, a CRM197 protein lyophilization solution is prepared containing of 6.0 ± 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized. In some embodiments, before conjugation, the lyophilized activated Pn15A saccharide and CRM197 protein are each reconstituted with anhydrous DMSO. In some embodiments, the lyophilized activated Pn15A saccharide is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL. In some embodiments, the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL. In some embodiments, after reconstitution, volumes of reconstituted activated Pn15A saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL, and the final concentration of CRM197 protein is about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL. In some embodiments, the combined solutions of reconstituted activated Pn15A saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ± 2°C, during which time the moisture level of the mixture can be monitored. In some embodiments, after the preincubation period, the conjugation reaction is initiated by adding to the mixture 0.75 to 1.25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn15A saccharide in the reaction mixture. In some embodiments, after the addition of the reducing agent, the conjugation reaction is incubated for 24 ± 4 hrs at 23 ± 2°C. In some embodiments, after the conjugation reaction has proceeded for the desired amount of time, the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH4. In some embodiments, after the addition of the capping agent, the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ± 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn15A saccharide-CRM ? protein conjugates is diluted and purified. In some embodiments, before conjugation, the activated Pn15A saccharide has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20-50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200-300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn15A saccharide has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16- 18, 17-18, or some other degree of oxidation or range. In some embodiments, after conjugation, the free saccharide content of Pn15A-CRM 7 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. In some embodiments, after conjugation, the yield of Pn15A-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, after conjugation, the average molecular weight (Mw) of Pn15A- CRM197 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250- 1750, 1500-2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250- 3750, 3500-4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn15A saccharide has Mw of about 75-225 kDa or about 100-200 kDa and DO of about 3-7 or about 4-7 or about 4-6. In some embodiments, after conjugation, the free saccharide content of Pn15A-CRM 7 conjugates is less than 10% and the yield is at least 60%. In some embodiments, after conjugation, the average Mw of Pn15A- CRM197 conjugates is about 1250-2750 kDa or about 1500-2500 kDa or about 1750-2250 kDa.
In some embodiments, bacterial capsular saccharide from Streptococcus pneumoniae serotype 23B (Pn23B) is activated and conjugated with CRM197 protein by reductive amination. In some embodiments, before conjugation, activated Pn23B saccharide and CRM197 protein are lyophilized separately. In some embodiments, before lyophilization, a CRM197 protein lyophilization solution is prepared containing of 6.0 ± 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized. In some embodiments, before conjugation, the lyophilized activated Pn23B saccharide and CRM197 protein are each reconstituted with anhydrous DMSO. In some embodiments, the lyophilized activated Pn23B saccharide is reconstituted with anhydrous DMSO to a concentration of about 5.0 to 7.0 mg/mL, or about 6.0 mg/mL. In some embodiments, the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 5.0 to 8.0 mg/mL, or about 6.6 mg/mL. In some embodiments, after reconstitution, volumes of reconstituted activated Pn23B saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide is about 2.5 to 3.5 mg/mL, or about 3.0 mg/mL, and the final concentration of CRM197 protein is about 2.5 to 4.0, or about 3.3 mg/mL. In some embodiments, the combined solutions of reconstituted activated Pn23B saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ± 2°C, during which time the moisture level of the mixture can be monitored. In some embodiments, after the preincubation period, the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn23B saccharide in the reaction mixture. In some embodiments, after the addition of the reducing agent, the conjugation reaction is incubated for 24 ± 4 hrs at 23 ± 2°C. In some embodiments, after the conjugation reaction has proceeded for the desired amount of time, the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH4. In some embodiments, after the addition of the capping agent, the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ± 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn23B saccharide-CRMi97 protein conjugates is diluted and purified. In some embodiments, before conjugation, the activated Pn23B saccharide has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20- 50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200- 300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn23B saccharide has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range. In some embodiments, after conjugation, the free saccharide content of Pn23B-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. In some embodiments, after conjugation, the yield of Pn23B-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, after conjugation, the average molecular weight (Mw) of Pn23B-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500- 2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500- 4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn23B saccharide has Mw of about 100-300 kDa or about 150-250 kDa and DO of about 7-11 or about 8-10. In some embodiments, after conjugation, the free saccharide content of Pn23B-CRMi97 conjugates is less than 10% and the yield is at least 50%. In some embodiments, after conjugation, the average Mw of Pn23B-CRMi97 conjugates is about 750-1500 or about 750-1250 kDa.
In some embodiments, bacterial capsular saccharide from Streptococcus pneumoniae serotype 35B (Pn35B) is activated and conjugated with CRM197 protein by reductive amination. In some embodiments, before conjugation, activated Pn35B saccharide and CRM197 protein are lyophilized separately. In some embodiments, before lyophilization, a CRM197 protein lyophilization solution is prepared containing of 6.0 ± 1.0 g/L CRM197 protein in 5 mM potassium phosphate buffer (pH 7.4) and 4.5% sucrose (w/v), which is then lyophilized. In some embodiments, before conjugation, the lyophilized activated Pn35B saccharide and CRM197 protein are each reconstituted with anhydrous DMSO. In some embodiments, the lyophilized activated Pn35B saccharide is reconstituted with anhydrous DMSO to a concentration of about 7.0 to 9.0 mg/mL, or about 8.0 mg/mL. In some embodiments, the lyophilized CRM197 protein is reconstituted with anhydrous DMSO to a concentration of about 8.0 to 12.0 mg/mL, or about 10.0 mg/mL. In some embodiments, after reconstitution, volumes of reconstituted activated Pn35B saccharide and CRM197 protein are combined in a reaction vessel such that the final concentration of activated saccharide about 3.5 to 4.5 mg/mL, or about 4.0 mg/mL, and the final concentration of CRM197 protein is about 4.0 to 6.0, or about 5.0 mg/mL. In some embodiments, the combined solutions of reconstituted activated Pn35B saccharide and CRM197 protein are preincubated by mixing for 60 - 120 minutes at 23 ± 2°C, during which time the moisture level of the mixture can be monitored. In some embodiments, after the preincubation period, the conjugation reaction is initiated by adding to the mixture 0.75 to 1 .25 molar equivalents of the reducing agent NaCNBHs, or about 1 .0 molar equivalents of NaCNBHs, relative to the amount of activated Pn35B saccharide in the reaction mixture. In some embodiments, after the addition of the reducing agent, the conjugation reaction is incubated for 24 ± 4 hrs at 23 ± 2°C. In some embodiments, after the conjugation reaction has proceeded for the desired amount of time, the conjugation reaction is terminated by addition of 1.75 to 2.25 molar equivalents of the capping agent NaBH4, or about 2.0 molar equivalents of NaBH4. In some embodiments, after the addition of the capping agent, the capping reaction is incubated for about 3-4 hours, or about 3 hours, at 23 ± 2°C. In some embodiments, after completing the conjugation reaction, the solution containing the Pn35B saccharide-CRMi97 protein conjugates is diluted and purified. In some embodiments, before conjugation, the activated Pn35B saccharide has an average molecular weight (Mw) of at least or about 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 kDa, such as, for example, 20- 50 kDa, 20-100 kDa, 50-100 kDa, 50-150 kDa, 100-150 kDa, 100-200 kDa, 150-250 kDa, 200- 300 kDa, 250-350 kDa, 300-400 kDa, 350-450 kDa, 400-500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn35B saccharide has degree of oxidation (DO) of about 3, 4, 5, 6, 7, 8, 9, 19, 11 , 12, 13, 14, 15, 16, 17, or 18, such as, for example 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7, 6-8, 7-8, 7-9, 8-9, 8-10, 9-10, 9-11 , 10-11 , 10-12, 11-12, 11-13, 12-13, 12-14, 13-14, 13-15, 14-15, 14-16, 15-16, 15-17, 16-17, 16-18, 17-18, or some other degree of oxidation or range. In some embodiments, after conjugation, the free saccharide content of Pn35B-CRMi97 conjugates is less than or about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. In some embodiments, after conjugation, the yield of Pn35B-CRMi97 conjugates is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, after conjugation, the average molecular weight (Mw) of Pn35B-CRM 7 conjugates is at least or about 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, or 4500, such as, for example, 750-1250, 1000-1500, 1250-1750, 1500- 2000, 1750-2250, 2000-2500, 2250-2750, 2500-3000, 2750-3250, 3000-3500, 3250-3750, 3500- 4000, 3750-4250, 4000-4500 kDa, or some other molecular weight or range. In some embodiments, before conjugation, the activated Pn35B saccharide has Mw of about 20-80 kDa or about 30-60 kDa or about 30-50 kDa and DO of about 6-10 or about 7-9. In some embodiments, after conjugation, the free saccharide content of Pn35B-CRMi97 conjugates is less than 10% and the yield is at least 55%. In some embodiments, after conjugation, the average Mw of Pn35B-CRMi97 conjugates is about 750-2500 or about 1000-2250 or about 1000-2000 kDa.
Purification after conjugation
In some embodiments, after the conjugation reaction, glycoconjugates can be purified, partially or fully, by a variety of techniques known to the skilled person. These techniques include, without limitation, dialysis, concentration/diafiltration operations against various buffers, tangential flow filtration precipitation/elution, column chromatography (DEAE or hydrophobic interaction chromatography, others being possible), membrane filtration, and depth filtration. Accordingly, in some embodiments, methods of the disclosure for producing a capsular saccharide glycoconjugate further comprise the step of purifying the glycoconjugate, partially or fully, after it is produced.
In some embodiments, a reaction mixture containing conjugates of saccharide and carrier protein can be diluted with a larger volume of a dilution solution. In some embodiments, the dilution solution can be a chilled 5 mM succinate-saline solution at pH 6.0, and the conjugation reaction mixture can be diluted with a 4x-9x volume of such dilution solution.
In some embodiments, after dilution, the diluted conjugation reaction mixture can be mixed for a period of time, such as 10 minutes, or some other time period, and then subjected to microfiltration, such as filtration through a 5 pm filter, or some other filter.
In some embodiments, after microfiltration, the diluted conjugation reaction mixture can be concentrated by diafiltration. In some embodiments, diafiltration can be performed against the same solution as the dilution solution. In some embodiments, the diafiltration solution can be a 5 mM succinate-saline solution at pH 6.0. In some embodiments, diafiltration is performed using a membrane filter with a certain molecular weight cutoff (MWCO), such as 100 kDa MWCO, or some other MWCO. In some embodiments, the membrane filter is a regenerated cellulose membrane filter. In some embodiments, diafiltration is performed under conditions where the membrane challenge is 1-10 grams saccharide per ft2 membrane area, transmembrane pressure is 5-20 PSI, and the retentate concentration is 1-5 g/L.
In some embodiments, after diafiltration, the concentrated conjugate solution is subjected to microfiltration, such as filtration through a 0.45 pm filter followed by filtration through a 0.22 pm filter, or some other filter.
Glycoconjugate molecular weight
After conjugation and purification, the weight average molecular weight (Mw) of glycoconjugates prepared by methods of the disclosure 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 glycoconjugates prepared by methods of the disclosure is at least or about 250 kilodaltons (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, 2100 kDa, 2200 kDa, 2300 kDa, 2400 kDa, 2500 kDa, 2600 kDa, 2700 kDa, 2800 kDa, 2900 kDa, 3000 kDa, 3100 kDa, 3200 kDa, 3300 kDa, 3400 kDa, 3500 kDa, 3600 kDa, 3700 kDa, 3800 kDa, 3900 kDa, 4000 kDa, 4100 kDa, 4200 kDa, 4300 kDa, 4400 kDa, 4500 kDa, 4600 kDa, 4700 kDa, 4800 kDa, 4900 kDa, 5000 kDa, 5500 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, 10000 kDa,
11000 kDa, 12000 kDa, 13000 kDa, 14000 kDa, 15000 kDa, 16000 kDa, 17000 kDa, 18000 kDa, 19000 kDa, 20000 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 glycoconjugates prepared by methods of the disclosure can range from about 250-20000 kDa, 250-15000 kDa, 250-10000 kDa, 250-9000 kDa, 250-8000 kDa, 250-7000 kDa, 250-6500 kDa, 250-6000 kDa, 250-5500 kDa, 250-5400 kDa, 250-5300 kDa, 250-5200 kDa, 250-5100 kDa, 250-5000 kDa, 250- 4900 kDa, 250-4800 kDa, 250-4700 kDa, 250-4600 kDa, 250-4500 kDa, 250-4400 kDa, 250-
4300 kDa, 250-4200 kDa, 250-4100 kDa, 250-4000 kDa, 250-3900 kDa, 250-3800 kDa, 250-
3700 kDa, 250-3600 kDa, 250-3500 kDa, 250-3400 kDa, 250-3300 kDa, 250-3200 kDa, 250-
3100 kDa, 250-3000 kDa, 250-2900 kDa, 250-2800 kDa, 250-2700 kDa, 250-2600 kDa, 250-
2500 kDa, 250-2400 kDa, 250-2300 kDa, 250-2200 kDa, 250-2100 kDa, 250-2000 kDa, 250-
1950 kDa, 250-1900 kDa, 250-1850 kDa, 250-1800 kDa, 250-1750 kDa, 250-1700 kDa, 250-
1650 kDa, 250-1600 kDa, 250-1550 kDa, 250-1500 kDa, 250-1450 kDa, 250-1400 kDa, 250-
1350 kDa, 250-1300 kDa, 250-1250 kDa, 250-1200 kDa, 250-1150 kDa, 250-1000 kDa, 250-950 kDa, 250-900 kDa, 250-850 kDa, 250-800 kDa, 250-750 kDa, 250-700 kDa, 250-650 kDa, 250- 600 kDa, 250-550 kDa, 250-500 kDa, 250-450 kDa, 250-400 kDa, 250-350 kDa, 250-300 kDa, 250-20000 kDa, 250-15000 kDa, 250-10000 kDa, 250-9000 kDa, 250-8000 kDa, 250-7000 kDa, 250-6500 kDa, 250-6000 kDa, 500-20000 kDa, 500-15000 kDa, 500-10000 kDa, 500-9000 kDa, 500-8000 kDa, 500-7000 kDa, 500-6500 kDa, 500-6000 kDa, 750-20000 kDa, 750-15000 kDa, 750-10000 kDa, 750-9000 kDa, 750-8000 kDa, 750-7000 kDa, 750-6500 kDa, 750-6000 kDa, 1000-20000 kDa, 1000-15000 kDa, 1000-10000 kDa, 1000-9000 kDa, 1000-8000 kDa, 1000- 7000 kDa, 1000-6500 kDa, 1000-6000 kDa, 1250-20000 kDa, 1250-15000 kDa, 1250-10000 kDa, 1250-9000 kDa, 1250-8000 kDa, 1250-7000 kDa, 1250-6500 kDa, 1250-6000 kDa, 1500- 20000 kDa, 1500-15000 kDa, 1500-10000 kDa, 1500-9000 kDa, 1500-8000 kDa, 1500-7000 kDa, 1500-6500 kDa, 1500-6000 kDa, 1750-20000 kDa, 1750-15000 kDa, 1750-10000 kDa, 1750-9000 kDa, 1750-8000 kDa, 1750-7000 kDa, 1750-6500 kDa, 1750-6000 kDa, 2000-20000 kDa, 2000-15000 kDa, 2000-10000 kDa, 2000-9000 kDa, 2000-8000 kDa, 2000-7000 kDa, 2000- 6500 kDa, 2000-6000 kDa, 2250-20000 kDa, 2250-15000 kDa, 2250-10000 kDa, 2250-9000 kDa, 2250-8000 kDa, 2250-7000 kDa, 2250-6500 kDa, 2250-6000 kDa, 2500-20000 kDa, 2500- 15000 kDa, 2500-10000 kDa, 2500-9000 kDa, 2500-8000 kDa, 2500-7000 kDa, 2500-6500 kDa, 2500-6000 kDa, 2750-20000 kDa, 2750-15000 kDa, 2750-10000 kDa, 2750-9000 kDa, 2750- 8000 kDa, 2750-7000 kDa, 2750-6500 kDa, 2750-6000 kDa, 3000-20000 kDa, 3000-15000 kDa, 3000-10000 kDa, 3000-9000 kDa, 3000-8000 kDa, 3000-7000 kDa, 3000-6500 kDa, 3000-6000 kDa, 3250-20000 kDa, 3250-15000 kDa, 3250-10000 kDa, 3250-9000 kDa, 3250-8000 kDa,
3250-7000 kDa, 3250-6500 kDa, 3250-6000 kDa, 3500-20000 kDa, 3500-15000 kDa, 3500-
10000 kDa, 3500-9000 kDa, 3500-8000 kDa, 3500-7000 kDa, 3500-6500 kDa, 3500-6000 kDa,
3750-20000 kDa, 3750-15000 kDa, 3750-10000 kDa, 3750-9000 kDa, 3750-8000 kDa, 3750-
7000 kDa, 3750-6500 kDa, 3750-6000 kDa, 4000-20000 kDa, 4000-15000 kDa, 4000-10000 kDa, 4000-9000 kDa, 4000-8000 kDa, 4000-7000 kDa, 4000-6500 kDa, 4000-6000 kDa, 4250-
20000 kDa, 4250-15000 kDa, 4250-10000 kDa, 4250-9000 kDa, 4250-8000 kDa, 4250-7000 kDa, 4250-6500 kDa, 4250-6000 kDa, 4500-20000 kDa, 4500-15000 kDa, 4500-10000 kDa,
4500-9000 kDa, 4500-8000 kDa, 4500-7000 kDa, 4500-6500 kDa, 4500-6000 kDa, 4750-20000 kDa, 4750-15000 kDa, 4750-10000 kDa, 4750-9000 kDa, 4750-8000 kDa, 4750-7000 kDa, 4750-
6500 kDa, 4750-6000 kDa, 5000-20000 kDa, 5000-15000 kDa, 5000-10000 kDa, 5000-9000 kDa, 5000-8000 kDa, 5000-7000 kDa, 5000-6500 kDa, 5000-6000 kDa, 5550-20000 kDa, 5550-
15000 kDa, 5550-10000 kDa, 5550-9000 kDa, 5550-8000 kDa, 5550-7000 kDa, 5550-6500 kDa,
5550-6000 kDa, 300-5000 kDa, 300-4900 kDa, 300-4800 kDa, 300-4700 kDa, 300-4600 kDa,
300-4500 kDa, 300-4400 kDa, 300-4300 kDa, 300-4200 kDa, 300-4100 kDa, 300-4000 kDa, 300-
3900 kDa, 300-3800 kDa, 300-3700 kDa, 300-3600 kDa, 300-3500 kDa, 300-3400 kDa, 300-
3300 kDa, 300-3200 kDa, 300-3100 kDa, 300-3000 kDa, 300-2900 kDa, 300-2800 kDa, 300-
2700 kDa, 300-2600 kDa, 300-2500 kDa, 300-2400 kDa, 300-2300 kDa, 300-2200 kDa, 300-
2100 kDa, 300-2000 kDa, 300-1950 kDa, 300-1900 kDa, 300-1850 kDa, 300-1800 kDa, 300-
1750 kDa, 300-1700 kDa, 300-1650 kDa, 300-1600 kDa, 300-1550 kDa, 300-1500 kDa, 300-
1450 kDa, 300-1400 kDa, 300-1350 kDa, 300-1300 kDa, 300-1250 kDa, 300-1200 kDa, 300-
1150 kDa, 300-1000 kDa, 300-950 kDa, 300-900 kDa, 300-850 kDa, 300-800 kDa, 300-750 kDa,
300-700 kDa, 300-650 kDa, 300-600 kDa, 300-550 kDa, 300-500 kDa, 300-450 kDa, 300-400 kDa, 300-350 kDa, 400-5000 kDa, 400-4900 kDa, 400-4800 kDa, 400-4700 kDa, 400-4600 kDa,
400-4500 kDa, 400-4400 kDa, 400-4300 kDa, 400-4200 kDa, 400-4100 kDa, 400-4000 kDa, 400-
3900 kDa, 400-3800 kDa, 400-3700 kDa, 400-3600 kDa, 400-3500 kDa, 400-3400 kDa, 400-
3300 kDa, 400-3200 kDa, 400-3100 kDa, 400-3000 kDa, 400-2900 kDa, 400-2800 kDa, 400-
2700 kDa, 400-2600 kDa, 400-2500 kDa, 400-2400 kDa, 400-2300 kDa, 400-2200 kDa, 400-
2100 kDa, 400-2000 kDa, 400-1950 kDa, 400-1900 kDa, 400-1850 kDa, 400-1800 kDa, 400-
1750 kDa, 400-1700 kDa, 400-1650 kDa, 400-1600 kDa, 400-1550 kDa, 400-1500 kDa, 400-
1450 kDa, 400-1400 kDa, 400-1350 kDa, 400-1300 kDa, 400-1250 kDa, 400-1200 kDa, 400-
1150 kDa, 400-1000 kDa, 400-950 kDa, 400-900 kDa, 400-850 kDa, 400-800 kDa, 400-750 kDa,
400-700 kDa, 400-650 kDa, 400-600 kDa, 400-550 kDa, 400-500 kDa, 400-450 kDa, 500-5000 kDa, 500-4900 kDa, 500-4800 kDa, 500-4700 kDa, 500-4600 kDa, 500-4500 kDa, 500-4400 kDa,
500-4300 kDa, 500-4200 kDa, 500-4100 kDa, 500-4000 kDa, 500-3900 kDa, 500-3800 kDa, 500-
3700 kDa, 500-3600 kDa, 500-3500 kDa, 500-3400 kDa, 500-3300 kDa, 500-3200 kDa, 500-
3100 kDa, 500-3000 kDa, 500-2900 kDa, 500-2800 kDa, 500-2700 kDa, 500-2600 kDa, 500- 2500 kDa, 500-2400 kDa, 500-2300 kDa, 500-2200 kDa, 500-2100 kDa, 500-2000 kDa, 500-
1950 kDa, 500-1900 kDa, 500-1850 kDa, 500-1800 kDa, 500-1750 kDa, 500-1700 kDa, 500-
1650 kDa, 500-1600 kDa, 500-1550 kDa, 500-1500 kDa, 500-1450 kDa, 500-1400 kDa, 500-
1350 kDa, 500-1300 kDa, 500-1250 kDa, 500-1200 kDa, 500-1150 kDa, 500-1000 kDa, 500-950 kDa, 500-900 kDa, 500-850 kDa, 500-800 kDa, 500-750 kDa, 500-700 kDa, 500-650 kDa, 500-
600 kDa, 500-550 kDa, 600-5000 kDa, 600-4900 kDa, 600-4800 kDa, 600-4700 kDa, 600-4600 kDa, 600-4500 kDa, 600-4400 kDa, 600-4300 kDa, 600-4200 kDa, 600-4100 kDa, 600-4000 kDa,
600-3900 kDa, 600-3800 kDa, 600-3700 kDa, 600-3600 kDa, 600-3500 kDa, 600-3400 kDa, 600-
3300 kDa, 600-3200 kDa, 600-3100 kDa, 600-3000 kDa, 600-2900 kDa, 600-2800 kDa, 600-
2700 kDa, 600-2600 kDa, 600-2500 kDa, 600-2400 kDa, 600-2300 kDa, 600-2200 kDa, 600-
2100 kDa, 600-2000 kDa, 600-1950 kDa, 600-1900 kDa, 600-1850 kDa, 600-1800 kDa, 600-
1750 kDa, 600-1700 kDa, 600-1650 kDa, 600-1600 kDa, 600-1550 kDa, 600-1500 kDa, 600-
1450 kDa, 600-1400 kDa, 600-1350 kDa, 600-1300 kDa, 600-1250 kDa, 600-1200 kDa, 600-
1150 kDa, 600-1000 kDa, 600-950 kDa, 600-900 kDa, 600-850 kDa, 600-800 kDa, 600-750 kDa,
600-700 kDa, 600-650 kDa, 700-5000 kDa, 700-4900 kDa, 700-4800 kDa, 700-4700 kDa, 700-
4600 kDa, 700-4500 kDa, 700-4400 kDa, 700-4300 kDa, 700-4200 kDa, 700-4100 kDa, 700-
4000 kDa, 700-3900 kDa, 700-3800 kDa, 700-3700 kDa, 700-3600 kDa, 700-3500 kDa, 700-
3400 kDa, 700-3300 kDa, 700-3200 kDa, 700-3100 kDa, 700-3000 kDa, 700-2900 kDa, 700-
2800 kDa, 700-2700 kDa, 700-2600 kDa, 700-2500 kDa, 700-2400 kDa, 700-2300 kDa, 700-
2200 kDa, 700-2100 kDa, 700-2000 kDa, 700-1950 kDa, 700-1900 kDa, 700-1850 kDa, 700-
1800 kDa, 700-1750 kDa, 700-1700 kDa, 700-1650 kDa, 700-1600 kDa, 700-1550 kDa, 700-
1500 kDa, 700-1450 kDa, 700-1400 kDa, 700-1350 kDa, 700-1300 kDa, 700-1250 kDa, 700-
1200 kDa, 700-1150 kDa, 700-1000 kDa, 700-950 kDa, 700-900 kDa, 700-850 kDa, 700-800 kDa, 700-750 kDa, 800-5000 kDa, 800-4900 kDa, 800-4800 kDa, 800-4700 kDa, 800-4600 kDa,
800-4500 kDa, 800-4400 kDa, 800-4300 kDa, 800-4200 kDa, 800-4100 kDa, 800-4000 kDa, 800-
3900 kDa, 800-3800 kDa, 800-3700 kDa, 800-3600 kDa, 800-3500 kDa, 800-3400 kDa, 800-
3300 kDa, 800-3200 kDa, 800-3100 kDa, 800-3000 kDa, 800-2900 kDa, 800-2800 kDa, 800-
2700 kDa, 800-2600 kDa, 800-2500 kDa, 800-2400 kDa, 800-2300 kDa, 800-2200 kDa, 800-
2100 kDa, 800-2000 kDa, 800-1950 kDa, 800-1900 kDa, 800-1850 kDa, 800-1800 kDa, 800-
1750 kDa, 800-1700 kDa, 800-1650 kDa, 800-1600 kDa, 800-1550 kDa, 800-1500 kDa, 800-
1450 kDa, 800-1400 kDa, 800-1350 kDa, 800-1300 kDa, 800-1250 kDa, 800-1200 kDa, 800-
1150 kDa, 800-1000 kDa, 800-950 kDa, 800-900 kDa, 800-850 kDa, 900-5000 kDa, 900-4900 kDa, 900-4800 kDa, 900-4700 kDa, 900-4600 kDa, 900-4500 kDa, 900-4400 kDa, 900-4300 kDa,
900-4200 kDa, 900-4100 kDa, 900-4000 kDa, 900-3900 kDa, 900-3800 kDa, 900-3700 kDa, 900-
3600 kDa, 900-3500 kDa, 900-3400 kDa, 900-3300 kDa, 900-3200 kDa, 900-3100 kDa, 900-
3000 kDa, 900-2900 kDa, 900-2800 kDa, 900-2700 kDa, 900-2600 kDa, 900-2500 kDa, 900-
2400 kDa, 900-2300 kDa, 900-2200 kDa, 900-2100 kDa, 900-2000 kDa, 900-1950 kDa, 900-
1900 kDa, 900-1850 kDa, 900-1800 kDa, 900-1750 kDa, 900-1700 kDa, 900-1650 kDa, 900- 1600 kDa, 900-1550 kDa, 900-1500 kDa, 900-1450 kDa, 900-1400 kDa, 900-1350 kDa, 900- 1300 kDa, 900-1250 kDa, 900-1200 kDa, 900-1150 kDa, 900-1000 kDa, 900-950 kDa, 1000- 5000 kDa, 1000-4900 kDa, 1000-4800 kDa, 1000-4700 kDa, 1000-4600 kDa, 1000-4500 kDa, 1000-4400 kDa, 1000-4300 kDa, 1000-4200 kDa, 1000-4100 kDa, 1000-4000 kDa, 1000-3900 kDa, 1000-3800 kDa, 1000-3700 kDa, 1000-3600 kDa, 1000-3500 kDa, 1000-3400 kDa, 1000- 3300 kDa, 1000-3200 kDa, 1000-3100 kDa, 1000-3000 kDa, 1000-2900 kDa, 1000-2800 kDa, 1000-2700 kDa, 1000-2600 kDa, 1000-2500 kDa, 1000-2400 kDa, 1000-2300 kDa, 1000-2200 kDa, 1000-2100 kDa, 1000-2000 kDa, 1000-1950 kDa, 1000-1900 kDa, 1000-1850 kDa, 1000- 1800 kDa, 1000-1750 kDa, 1000-1700 kDa, 1000-1650 kDa, 1000-1600 kDa, 1000-1550 kDa, 1000-1500 kDa, 1000-1450 kDa, 1000-1400 kDa, 1000-1350 kDa, 1000-1300 kDa, 1000-1250 kDa, 1000-1200 kDa, 1000-1150 kDa, 1100-5000 kDa, 1100-4900 kDa, 1100-4800 kDa, 1100- 4700 kDa, 1100-4600 kDa, 1100-4500 kDa, 1100-4400 kDa, 1100-4300 kDa, 1100-4200 kDa, 1100-4100 kDa, 1100-4000 kDa, 1100-3900 kDa, 1100-3800 kDa, 1100-3700 kDa, 1100-3600 kDa, 1100-3500 kDa, 1100-3400 kDa, 1100-3300 kDa, 1100-3200 kDa, 1100-3100 kDa, 1100- 3000 kDa, 1100-2900 kDa, 1100-2800 kDa, 1100-2700 kDa, 1100-2600 kDa, 1100-2500 kDa, 1100-2400 kDa, 1100-2300 kDa, 1100-2200 kDa, 1100-2100 kDa, 1100-2000 kDa, 1100-1950 kDa, 1100-1900 kDa, 1100-1850 kDa, 1100-1800 kDa, 1100-1750 kDa, 1100-1700 kDa, 1100- 1650 kDa, 1100-1600 kDa, 1100-1550 kDa, 1100-1500 kDa, 1100-1450 kDa, 1100-1400 kDa, 1100-1350 kDa, 1100-1300 kDa, 1100-1250 kDa, 1100-1200 kDa, 1100-1150 kDa, 1200-5000 kDa, 1200-4900 kDa, 1200-4800 kDa, 1200-4700 kDa, 1200-4600 kDa, 1200-4500 kDa, 1200- 4400 kDa, 1200-4300 kDa, 1200-4200 kDa, 1200-4100 kDa, 1200-4000 kDa, 1200-3900 kDa, 1200-3800 kDa, 1200-3700 kDa, 1200-3600 kDa, 1200-3500 kDa, 1200-3400 kDa, 1200-3300 kDa, 1200-3200 kDa, 1200-3100 kDa, 1200-3000 kDa, 1200-2900 kDa, 1200-2800 kDa, 1200- 2700 kDa, 1200-2600 kDa, 1200-2500 kDa, 1200-2400 kDa, 1200-2300 kDa, 1200-2200 kDa, 1200-2100 kDa, 1200-2000 kDa, 1200-1950 kDa, 1200-1900 kDa, 1200-1850 kDa, 1200-1800 kDa, 1200-1750 kDa, 1200-1700 kDa, 1200-1650 kDa, 1200-1600 kDa, 1200-1550 kDa, 1200- 1500 kDa, 1200-1450 kDa, 1200-1400 kDa, 1200-1350 kDa, 1200-1300 kDa, 1200-1250 kDa, 1300-5000 kDa, 1300-4900 kDa, 1300-4800 kDa, 1300-4700 kDa, 1300-4600 kDa, 1300-4500 kDa, 1300-4400 kDa, 1300-4300 kDa, 1300-4200 kDa, 1300-4100 kDa, 1300-4000 kDa, 1300- 3900 kDa, 1300-3800 kDa, 1300-3700 kDa, 1300-3600 kDa, 1300-3500 kDa, 1300-3400 kDa, 1300-3300 kDa, 1300-3200 kDa, 1300-3100 kDa, 1300-3000 kDa, 1300-2900 kDa, 1300-2800 kDa, 1300-2700 kDa, 1300-2600 kDa, 1300-2500 kDa, 1300-2400 kDa, 1300-2300 kDa, 1300- 2200 kDa, 1300-2100 kDa, 1300-2000 kDa, 1300-1950 kDa, 1300-1900 kDa, 1300-1850 kDa, 1300-1800 kDa, 1300-1750 kDa, 1300-1700 kDa, 1300-1650 kDa, 1300-1600 kDa, 1300-1550 kDa, 1300-1500 kDa, 1300-1450 kDa, 1300-1400 kDa, 1300-1350 kDa, 1400-5000 kDa, 1400- 4900 kDa, 1400-4800 kDa, 1400-4700 kDa, 1400-4600 kDa, 1400-4500 kDa, 1400-4400 kDa, 1400-4300 kDa, 1400-4200 kDa, 1400-4100 kDa, 1400-4000 kDa, 1400-3900 kDa, 1400-3800 kDa, 1400-3700 kDa, 1400-3600 kDa, 1400-3500 kDa, 1400-3400 kDa, 1400-3300 kDa, 1400- 3200 kDa, 1400-3100 kDa, 1400-3000 kDa, 1400-2900 kDa, 1400-2800 kDa, 1400-2700 kDa, 1400-2600 kDa, 1400-2500 kDa, 1400-2400 kDa, 1400-2300 kDa, 1400-2200 kDa, 1400-2100 kDa, 1400-2000 kDa, 1400-1950 kDa, 1400-1900 kDa, 1400-1850 kDa, 1400-1800 kDa, 1400- 1750 kDa, 1400-1700 kDa, 1400-1650 kDa, 1400-1600 kDa, 1400-1550 kDa, 1400-1500 kDa, 1400-1450 kDa, 1500-5000 kDa, 1500-4900 kDa, 1500-4800 kDa, 1500-4700 kDa, 1500-4600 kDa, 1500-4500 kDa, 1500-4400 kDa, 1500-4300 kDa, 1500-4200 kDa, 1500-4100 kDa, 1500- 4000 kDa, 1500-3900 kDa, 1500-3800 kDa, 1500-3700 kDa, 1500-3600 kDa, 1500-3500 kDa, 1500-3400 kDa, 1500-3300 kDa, 1500-3200 kDa, 1500-3100 kDa, 1500-3000 kDa, 1500-2900 kDa, 1500-2800 kDa, 1500-2700 kDa, 1500-2600 kDa, 1500-2500 kDa, 1500-2400 kDa, 1500- 2300 kDa, 1500-2200 kDa, 1500-2100 kDa, 1500-2000 kDa, 1500-1950 kDa, 1500-1900 kDa, 1500-1850 kDa, 1500-1800 kDa, 1500-1750 kDa, 1500-1700 kDa, 1500-1650 kDa, 1500-1600 kDa, 1500-1550 kDa, 1600-5000 kDa, 1600-4900 kDa, 1600-4800 kDa, 1600-4700 kDa, 1600- 4600 kDa, 1600-4500 kDa, 1600-4400 kDa, 1600-4300 kDa, 1600-4200 kDa, 1600-4100 kDa, 1600-4000 kDa, 1600-3900 kDa, 1600-3800 kDa, 1600-3700 kDa, 1600-3600 kDa, 1600-3500 kDa, 1600-3400 kDa, 1600-3300 kDa, 1600-3200 kDa, 1600-3100 kDa, 1600-3000 kDa, 1600- 2900 kDa, 1600-2800 kDa, 1600-2700 kDa, 1600-2600 kDa, 1600-2500 kDa, 1600-2400 kDa, 1600-2300 kDa, 1600-2200 kDa, 1600-2100 kDa, 1600-2000 kDa, 1600-1950 kDa, 1600-1900 kDa, 1600-1850 kDa, 1600-1800 kDa, 1600-1750 kDa, 1600-1700 kDa, 1600-1650 kDa, 1700- 5000 kDa, 1700-4900 kDa, 1700-4800 kDa, 1700-4700 kDa, 1700-4600 kDa, 1700-4500 kDa, 1700-4400 kDa, 1700-4300 kDa, 1700-4200 kDa, 1700-4100 kDa, 1700-4000 kDa, 1700-3900 kDa, 1700-3800 kDa, 1700-3700 kDa, 1700-3600 kDa, 1700-3500 kDa, 1700-3400 kDa, 1700- 3300 kDa, 1700-3200 kDa, 1700-3100 kDa, 1700-3000 kDa, 1700-2900 kDa, 1700-2800 kDa, 1700-2700 kDa, 1700-2600 kDa, 1700-2500 kDa, 1700-2400 kDa, 1700-2300 kDa, 1700-2200 kDa, 1700-2100 kDa, 1700-2000 kDa, 1700-1950 kDa, 1700-1900 kDa, 1700-1850 kDa, 1700- 1800 kDa, 1700-1750 kDa, 1800-5000 kDa, 1800-4900 kDa, 1800-4800 kDa, 1800-4700 kDa, 1800-4600 kDa, 1800-4500 kDa, 1800-4400 kDa, 1800-4300 kDa, 1800-4200 kDa, 1800-4100 kDa, 1800-4000 kDa, 1800-3900 kDa, 1800-3800 kDa, 1800-3700 kDa, 1800-3600 kDa, 1800- 3500 kDa, 1800-3400 kDa, 1800-3300 kDa, 1800-3200 kDa, 1800-3100 kDa, 1800-3000 kDa, 1800-2900 kDa, 1800-2800 kDa, 1800-2700 kDa, 1800-2600 kDa, 1800-2500 kDa, 1800-2400 kDa, 1800-2300 kDa, 1800-2200 kDa, 1800-2100 kDa, 1800-2000 kDa, 1800-1950 kDa, 1800- 1900 kDa, 1800-1850 kDa, 1900-5000 kDa, 1900-4900 kDa, 1900-4800 kDa, 1900-4700 kDa, 1900-4600 kDa, 1900-4500 kDa, 1900-4400 kDa, 1900-4300 kDa, 1900-4200 kDa, 1900-4100 kDa, 1900-4000 kDa, 1900-3900 kDa, 1900-3800 kDa, 1900-3700 kDa, 1900-3600 kDa, 1900- 3500 kDa, 1900-3400 kDa, 1900-3300 kDa, 1900-3200 kDa, 1900-3100 kDa, 1900-3000 kDa, 1900-2900 kDa, 1900-2800 kDa, 1900-2700 kDa, 1900-2600 kDa, 1900-2500 kDa, 1900-2400 kDa, 1900-2300 kDa, 1900-2200 kDa, 1900-2100 kDa, 1900-2000 kDa, 1900-1950 kDa, 2000- 5000 kDa, 2000-4900 kDa, 2000-4800 kDa, 2000-4700 kDa, 2000-4600 kDa, 2000-4500 kDa, 2000-4400 kDa, 2000-4300 kDa, 2000-4200 kDa, 2000-4100 kDa, 2000-4000 kDa, 2000-3900 kDa, 2000-3800 kDa, 2000-3700 kDa, 2000-3600 kDa, 2000-3500 kDa, 2000-3400 kDa, 2000- 3300 kDa, 2000-3200 kDa, 2000-3100 kDa, 2000-3000 kDa, 2000-2900 kDa, 2000-2800 kDa, 2000-2700 kDa, 2000-2600 kDa, 2000-2500 kDa, 2000-2400 kDa, 2000-2300 kDa, 2000-2200 kDa, 2000-2100 kDa, 2100-5000 kDa, 2100-4900 kDa, 2100-4800 kDa, 2100-4700 kDa, 2100- 4600 kDa, 2100-4500 kDa, 2100-4400 kDa, 2100-4300 kDa, 2100-4200 kDa, 2100-4100 kDa, 2100-4000 kDa, 2100-3900 kDa, 2100-3800 kDa, 2100-3700 kDa, 2100-3600 kDa, 2100-3500 kDa, 2100-3400 kDa, 2100-3300 kDa, 2100-3200 kDa, 2100-3100 kDa, 2100-3000 kDa, 2100- 2900 kDa, 2100-2800 kDa, 2100-2700 kDa, 2100-2600 kDa, 2100-2500 kDa, 2100-2400 kDa, 2100-2300 kDa, 2100-2200 kDa, 2200-5000 kDa, 2200-4900 kDa, 2200-4800 kDa, 2200-4700 kDa, 2200-4600 kDa, 2200-4500 kDa, 2200-4400 kDa, 2200-4300 kDa, 2200-4200 kDa, 2200- 4100 kDa, 2200-4000 kDa, 2200-3900 kDa, 2200-3800 kDa, 2200-3700 kDa, 2200-3600 kDa, 2200-3500 kDa, 2200-3400 kDa, 2200-3300 kDa, 2200-3200 kDa, 2200-3100 kDa, 2200-3000 kDa, 2200-2900 kDa, 2200-2800 kDa, 2200-2700 kDa, 2200-2600 kDa, 2200-2500 kDa, 2200- 2400 kDa, 2200-2300 kDa, 2300-5000 kDa, 2300-4900 kDa, 2300-4800 kDa, 2300-4700 kDa, 2300-4600 kDa, 2300-4500 kDa, 2300-4400 kDa, 2300-4300 kDa, 2300-4200 kDa, 2300-4100 kDa, 2300-4000 kDa, 2300-3900 kDa, 2300-3800 kDa, 2300-3700 kDa, 2300-3600 kDa, 2300- 3500 kDa, 2300-3400 kDa, 2300-3300 kDa, 2300-3200 kDa, 2300-3100 kDa, 2300-3000 kDa, 2300-2900 kDa, 2300-2800 kDa, 2300-2700 kDa, 2300-2600 kDa, 2300-2500 kDa, 2300-2400 kDa, 2400-5000 kDa, 2400-4900 kDa, 2400-4800 kDa, 2400-4700 kDa, 2400-4600 kDa, 2400- 4500 kDa, 2400-4400 kDa, 2400-4300 kDa, 2400-4200 kDa, 2400-4100 kDa, 2400-4000 kDa, 2400-3900 kDa, 2400-3800 kDa, 2400-3700 kDa, 2400-3600 kDa, 2400-3500 kDa, 2400-3400 kDa, 2400-3300 kDa, 2400-3200 kDa, 2400-3100 kDa, 2400-3000 kDa, 2400-2900 kDa, 2400- 2800 kDa, 2400-2700 kDa, 2400-2600 kDa, 2400-2500 kDa, 2400-2400 kDa, 2500-5000 kDa, 2500-4900 kDa, 2500-4800 kDa, 2500-4700 kDa, 2500-4600 kDa, 2500-4500 kDa, 2500-4400 kDa, 2500-4300 kDa, 2500-4200 kDa, 2500-4100 kDa, 2500-4000 kDa, 2500-3900 kDa, 2500- 3800 kDa, 2500-3700 kDa, 2500-3600 kDa, 2500-3500 kDa, 2500-3400 kDa, 2500-3300 kDa, 2500-3200 kDa, 2500-3100 kDa, 2500-3000 kDa, 2500-2900 kDa, 2500-2800 kDa, 2500-2700 kDa, 2500-2600 kDa, 2600-5000 kDa, 2600-4900 kDa, 2600-4800 kDa, 2600-4700 kDa, 2600- 4600 kDa, 2600-4500 kDa, 2600-4400 kDa, 2600-4300 kDa, 2600-4200 kDa, 2600-4100 kDa, 2600-4000 kDa, 2600-3900 kDa, 2600-3800 kDa, 2600-3700 kDa, 2600-3600 kDa, 2600-3500 kDa, 2600-3400 kDa, 2600-3300 kDa, 2600-3200 kDa, 2600-3100 kDa, 2600-3000 kDa, 2600- 2900 kDa, 2600-2800 kDa, 2600-2700 kDa, 2700-5000 kDa, 2700-4900 kDa, 2700-4800 kDa, 2700-4700 kDa, 2700-4600 kDa, 2700-4500 kDa, 2700-4400 kDa, 2700-4300 kDa, 2700-4200 kDa, 2700-4100 kDa, 2700-4000 kDa, 2700-3900 kDa, 2700-3800 kDa, 2700-3700 kDa, 2700- 3600 kDa, 2700-3500 kDa, 2700-3400 kDa, 2700-3300 kDa, 2700-3200 kDa, 2700-3100 kDa, 2700-3000 kDa, 2700-2900 kDa, 2700-2800 kDa, 2800-5000 kDa, 2800-4900 kDa, 2800-4800 kDa, 2800-4700 kDa, 2800-4600 kDa, 2800-4500 kDa, 2800-4400 kDa, 2800-4300 kDa, 2800- 4200 kDa, 2800-4100 kDa, 2800-4000 kDa, 2800-3900 kDa, 2800-3800 kDa, 2800-3700 kDa, 2800-3600 kDa, 2800-3500 kDa, 2800-3400 kDa, 2800-3300 kDa, 2800-3200 kDa, 2800-3100 kDa, 2800-3000 kDa, 2800-2900 kDa, 2900-5000 kDa, 2900-4900 kDa, 2900-4800 kDa, 2900- 4700 kDa, 2900-4600 kDa, 2900-4500 kDa, 2900-4400 kDa, 2900-4300 kDa, 2900-4200 kDa, 2900-4100 kDa, 2900-4000 kDa, 2900-3900 kDa, 2900-3800 kDa, 2900-3700 kDa, 2900-3600 kDa, 2900-3500 kDa, 2900-3400 kDa, 2900-3300 kDa, 2900-3200 kDa, 2900-3100 kDa, 2900- 3000 kDa, 3000-5000 kDa, 3000-4900 kDa, 3000-4800 kDa, 3000-4700 kDa, 3000-4600 kDa, 3000-4500 kDa, 3000-4400 kDa, 3000-4300 kDa, 3000-4200 kDa, 3000-4100 kDa, 3000-4000 kDa, 3000-3900 kDa, 3000-3800 kDa, 3000-3700 kDa, 3000-3600 kDa, 3000-3500 kDa, 3000- 3400 kDa, 3000-3300 kDa, 3000-3200 kDa, 3000-3100 kDa, 3100-5000 kDa, 3100-4900 kDa, 3100-4800 kDa, 3100-4700 kDa, 3100-4600 kDa, 3100-4500 kDa, 3100-4400 kDa, 3100-4300 kDa, 3100-4200 kDa, 3100-4100 kDa, 3100-4000 kDa, 3100-3900 kDa, 3100-3800 kDa, 3100- 3700 kDa, 3100-3600 kDa, 3100-3500 kDa, 3100-3400 kDa, 3100-3300 kDa, 3100-3200 kDa, 3200-5000 kDa, 3200-4900 kDa, 3200-4800 kDa, 3200-4700 kDa, 3200-4600 kDa, 3200-4500 kDa, 3200-4400 kDa, 3200-4300 kDa, 3200-4200 kDa, 3200-4100 kDa, 3200-4000 kDa, 3200- 3900 kDa, 3200-3800 kDa, 3200-3700 kDa, 3200-3600 kDa, 3200-3500 kDa, 3200-3400 kDa, 3200-3300 kDa, 3300-5000 kDa, 3300-4900 kDa, 3300-4800 kDa, 3300-4700 kDa, 3300-4600 kDa, 3300-4500 kDa, 3300-4400 kDa, 3300-4300 kDa, 3300-4200 kDa, 3300-4100 kDa, 3300- 4000 kDa, 3300-3900 kDa, 3300-3800 kDa, 3300-3700 kDa, 3300-3600 kDa, 3300-3500 kDa, 3300-3400 kDa, 3400-5000 kDa, 3400-4900 kDa, 3400-4800 kDa, 3400-4700 kDa, 3400-4600 kDa, 3400-4500 kDa, 3400-4400 kDa, 3400-4300 kDa, 3400-4200 kDa, 3400-4100 kDa, 3400- 4000 kDa, 3400-3900 kDa, 3400-3800 kDa, 3400-3700 kDa, 3400-3600 kDa, 3400-3500 kDa, 3500-5000 kDa, 3500-4900 kDa, 3500-4800 kDa, 3500-4700 kDa, 3500-4600 kDa, 3500-4500 kDa, 3500-4400 kDa, 3500-4300 kDa, 3500-4200 kDa, 3500-4100 kDa, 3500-4000 kDa, 3500- 3900 kDa, 3500-3800 kDa, 3500-3700 kDa, 3500-3600 kDa, 3600-5000 kDa, 3600-4900 kDa, 3600-4800 kDa, 3600-4700 kDa, 3600-4600 kDa, 3600-4500 kDa, 3600-4400 kDa, 3600-4300 kDa, 3600-4200 kDa, 3600-4100 kDa, 3600-4000 kDa, 3600-3900 kDa, 3600-3800 kDa, 3600- 3700 kDa, 3700-5000 kDa, 3700-4900 kDa, 3700-4800 kDa, 3700-4700 kDa, 3700-4600 kDa, 3700-4500 kDa, 3700-4400 kDa, 3700-4300 kDa, 3700-4200 kDa, 3700-4100 kDa, 3700-4000 kDa, 3700-3900 kDa, 3700-3800 kDa, 3800-5000 kDa, 3800-4900 kDa, 3800-4800 kDa, 3800- 4700 kDa, 3800-4600 kDa, 3800-4500 kDa, 3800-4400 kDa, 3800-4300 kDa, 3800-4200 kDa, 3800-4100 kDa, 3800-4000 kDa, 3800-3900 kDa, 3900-5000 kDa, 3900-4900 kDa, 3900-4800 kDa, 3900-4700 kDa, 3900-4600 kDa, 3900-4500 kDa, 3900-4400 kDa, 3900-4300 kDa, 3900- 4200 kDa, 3900-4100 kDa, 3900-4000 kDa, 4000-5000 kDa, 4000-4900 kDa, 4000-4800 kDa, 4000-4700 kDa, 4000-4600 kDa, 4000-4500 kDa, 4000-4400 kDa, 4000-4300 kDa, 4000-4200 kDa, 4000-4100 kDa, 4100-5000 kDa, 4100-4900 kDa, 4100-4800 kDa, 4100-4700 kDa, 4100- 4600 kDa, 4100-4500 kDa, 4100-4400 kDa, 4100-4300 kDa, 4100-4200 kDa, 4200-5000 kDa, 4200-4900 kDa, 4200-4800 kDa, 4200-4700 kDa, 4200-4600 kDa, 4200-4500 kDa, 4200-4400 kDa, 4200-4300 kDa, 4300-5000 kDa, 4300-4900 kDa, 4300-4800 kDa, 4300-4700 kDa, 4300- 4600 kDa, 4300-4500 kDa, 4300-4400 kDa, 4400-5000 kDa, 4400-4900 kDa, 4400-4800 kDa, 4400-4700 kDa, 4400-4600 kDa, 4400-4500 kDa, 4500-5000 kDa, 4500-4900 kDa, 4500-4800 kDa, 4500-4700 kDa, 4500-4600 kDa, 4600-5000 kDa, 4600-4900 kDa, 4600-4800 kDa, 4600- 4700 kDa, 4700-5000 kDa, 4700-4900 kDa, 4700-4800 kDa, 4800-5000 kDa, 4800-4900 kDa, or 4900-5000 kDa, or some other range.
Glycoconjugate degree of conjugation of carrier protein
In some embodiments, after purification, glycoconjugates of the disclosure can be characterized by the degree of conjugation of the carrier protein, which is the average number of lysine residues in the carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) that are conjugated to saccharide in a sample or preparation of glycoconjugate. Degree of conjugation of carrier protein of a glycoconjugate can be determined by amino acid analysis using routine methods known to those of skill in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate materials.
In some embodiments, the degree of conjugation of carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in glycoconjugates 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. For example, in some non-limiting embodiments, the degree of conjugation of carrier protein in glycoconjugates can be about 2-20,
2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11 , 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20,
3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11 , 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19,
4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5- 17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11 , 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11 , 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7- 11 , 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11 , 8-10, 8-9, 9-20, 9- 19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11 , 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, IQ- 15, 10-14, 10-13, 10-12, 10-11 , 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13- 15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 16-16, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20, or some other degree of conjugation. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197.
Glycoconjugate saccharide to carrier protein weight ratio
In some embodiments, after purification, glycoconjugates of the disclosure can be characterized by the ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of a glycoconjugate on a weight to weight (w/w) basis. The ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of a glycoconjugate can be determined using routine methods known to those of skill in the art. In some embodiments, the ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of glycoconjugate on a weight by weight basis is at least or about 0.5, 0.6, 0.7, 0.8, 0.9, 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, 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 ratio of saccharide to carrier protein (e.g., e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197) in a sample or preparation of glycoconjugate on a weight by weight basis can be about 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.5-1.0, 0.5-1.1 , 0.5-1.2, 0.5-1.3, 0.5-1.4, 0.5-1.5, 0.5-1.6, 0.5-1.7, 0.5-1.8, 0.5-1.9, 0.5-2.0, 0.5-2.1 , 0.5-2.2, 0.5-2.3, 0.5-2.4, 0.5-
2.5, 0.5-2.6, 0.5-2.7, 0.5-2.8, 0.5-2.9, 0.5-3.0, 0.6-0.7, 0.6-0.8, 0.6-0.9, 0.6-1.0, 0.6-1.1 , 0.6-1.2, 0.6-1.3, 0.6-1.4, 0.6-1.5, 0.6-1.6, 0.6-1.7, 0.6-1.8, 0.6-1.9, 0.6-2.0, 0.6-2.1 , 0.6-2.2, 0.6-2.3, 0.6- 2.4, 0.6-2.5, 0.6-2.6, 0.6-2.7, 0.6-2.8, 0.6-2.9, 0.6-3.0, 0.7-0.8, 0.7-0.9, 0.7-1.0, 0.7-1.1 , 0.7-1.2, 0.7-1.3, 0.7-1.4, 0.7-1.5, 0.7-1.6, 0.7-1.7, 0.7-1.8, 0.7-1.9, 0.7-2.0, 0.7-2.1 , 0.7-2.2, 0.7-2.3, 0.7-
2.4, 0.7-2.5, 0.7-2.6, 0.7-2.7, 0.7-2.8, 0.7-2.9, 0.7-3.0, 0.8-0.9, 0.8-1.0, 0.8-1.1 , 0.8-1.2, 0.8-1.3, 0.8-1.4, 0.8-1.5, 0.8-1.6, 0.8-1.7, 0.8-1.8, 0.8-1.9, 0.8-2.0, 0.8-2.1 , 0.8-2.2, 0.8-2.3, 0.8-2.4, 0.8-
2.5, 0.8-2.6, 0.8-2.7, 0.8-2.8, 0.8-2.9, 0.8-3.0, 0.9-1.0, 0.9-1.1 , 0.9-1.2, 0.9-1.3, 0.9-1.4, 0.9-1.5, 0.9-1.6, 0.9-1.7, 0.9-1.8, 0.9-1.9, 0.9-2.0, 0.9-2.1 , 0.9-2.2, 0.9-2.3, 0.9-2.4, 0.9-2.5, 0.9-2.6, 0.9- 2.7, 0.9-2.8, 0.9-2.9, 0.9-3.0, 1.0-1.1 , 1.0-1.2, 1.0-1.3, 1.0-1.4, 1.0-1.5, 1.0-1.6, 1.0-1.7, 1.0-1.8, 1.0-1.9, 1.0-2.0, 1.0-2.1 , 1.0-2.2, 1.0-2.3, 1.0-2.4, 1.0-2.5, 1.0-2.6, 1.0-2.7, 1.0-2.8, 1.0-2.9, 1.0- 3.0, 1.1-1.2, 1.1-1.3, 1.1-1.4, 1.1-1.5, 1.1-1.6, 1.1-1.7, 1.1-1.8, 1.1-1.9, 1.1-2.0, 1.1-2.1 , 1.1-2.2,
1.1-2.3, 1.1-2.4, 1.1-2.5, 1.1-2.6, 1.1-2.7, 1.1-2.8, 1.1-2.9, 1.1-3.0, 1.2-1.3, 1.2-1.4, 1.2-1.5, 1.2-
1.6, 1.2-1.7, 1.2-1.8, 1.2-1.9, 1.2-2.0, 1.2-2.1 , 1.2-2.2, 1.2-2.3, 1.2-2.4, 1.2-2.5, 1.2-2.6, 1.2-2.7,
1.2-2.8, 1.2-2.9, 1.2-3.0, 1.3-1.4, 1.3-1.5, 1.3-1.6, 1.3-1.7, 1.3-1.8, 1.3-1.9, 1.3-2.0, 1.3-2.1 , 1.3-
2.2, 1.3-2.3, 1.3-2.4, 1.3-2.5, 1.3-2.6, 1.3-2.7, 1.3-2.8, 1.3-2.9, 1.3-3.0, 1.4-1.5, 1.4-1.6, 1.4-1.7,
1.4-1.8, 1.4-1.9, 1.4-2.0, 1.4-2.1 , 1.4-2.2, 1.4-2.3, 1.4-2.4, 1.4-2.5, 1.4-2.6, 1.4-2.7, 1.4-2.8, 1.4- 2.9, 1.4-3.0, 1.5-1.6, 1.5-1.7, 1.5-1.8, 1.5-1.9, 1.5-2.0, 1.5-2.1 , 1.5-2.2, 1.5-2.3, 1.5-2.4, 1.5-2.5,
1.5-2.6, 1.5-2.7, 1.5-2.8, 1.5-2.9, 1.5-3.0, 1.6-1.7, 1.6-1.8, 1.6-1.9, 1.6-2.0, 1.6-2.1 , 1.6-2.2, 1.6-
2.3, 1.6-2.4, 1.6-2.5, 1.6-2.6, 1.6-2.7, 1.6-2.8, 1.6-2.9, 1.6-3.0, 1.7-1.8, 1.7-1.9, 1.7-2.0, 1.7-2.1 , 1.7-2.2, 1.7-2.3, 1.7-2.4, 1.7-2.5, 1.7-2.6, 1.7-2.7, 1.7-2.8, 1.7-2.9, 1.7-3.0, 1.8-1.9, 1.8-2.0, 1.8- 2.1 , 1.8-2.2, 1.8-2.3, 1.8-2.4, 1.8-2.5, 1.8-2.6, 1.8-2.7, 1.8-2.8, 1.8-2.9, 1.8-3.0, 1.9-2.0, 1.9-2.1 , 1.9-2.2, 1.9-2.3, 1.9-2.4, 1.9-2.5, 1.9-2.6, 1.9-2.7, 1.9-2.8, 1.9-2.9, 1.9-3.0, 2.0-2.1 , 2.0-2.2, 2.0-
2.3, 2.0-2.4, 2.0-2.5, 2.0-2.6, 2.0-2.7, 2.0-2.8, 2.0-2.9, 2.0-3.0, 2.1-2.2, 2.1-2.3, 2.1-2.4, 2.1-2.5, 2.1-2.6, 2.1-2.7, 2.1-2.8, 2.1-2.9, 2.1-3.0, 2.2-2.3, 2.2-2.4, 2.2-2.5, 2.2-2.6, 2.2-2.7, 2.2-2.8, 2.2- 2.9, 2.2-3.0, 2.3-2.4, 2.3-2.5, 2.3-2.6, 2.3-2.7, 2.3-2.8, 2.3-2.9, 2.3-3.0, 2.4-2.5, 2.4-2.6, 2.4-2.7, 2.4-2.8, 2.4-2.9, 2.4-3.0, 2.5-2.6, 2.5-2.7, 2.5-2.8, 2.5-2.9, 2.5-3.0, 2.6-2.7, 2.6-2.8, 2.6-2.9, 2.6- 3.0, 2.7-2.8, 2.7-2.9, 2.7-3.0, 2.8-2.9, 2.8-3.0, 2.9-3.0, or some other range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197.
Glycoconjugate free saccharide content
In some embodiments, after purification, glycoconjugates of the disclosure can be characterized by the amount of saccharide in a sample or preparation of a glycoconjugate which is free on a percent basis. The amount of saccharide in a sample or preparation of a glycoconjugate which is free can be determined using routine methods known to those of skill in the art. In some embodiments, the percentage of saccharide in a sample or preparation of a glycoconjugate which is free saccharide is less than or about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.
Glycoconjugate carrier protein-saccharide covalent linkages per repeat unit
In some embodiments, glycoconjugates of the disclosure can be characterized by the number of covalent linkages between the carrier protein and the saccharide as a function of the number of repeat units of the saccharide. As understood in the art, a saccharide repeat unit may comprise one or more monosaccharides of same or different type which are covalently linked in a consistent way to form a structural unit which in turn is covalently linked in a consistent way to others of the same type repeatedly to form the saccharide polymer. Saccharide repeat units may also comprise chemical groups other than monosaccharides, for examples, phosphate groups, other groups being possible. Capsular saccharides can be distinguished both by differences among the sizes, constituents, and structures of their respective repeat units, and/or in the way in which repeat units are linked to each other forming the saccharide.
In some embodiments, glycoconjugates can comprise at least 1 covalent linkage between the carrier protein and the saccharide per every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or more saccharide repeat units of the saccharide, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range. In any of the foregoing embodiments, the carrier protein can be DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197.
Glycoconjugate molecular size distribution
In some embodiments, glycoconjugates of the disclosure can be characterized by their molecular size distribution (Kd), which can be determined in a variety of ways familiar to those of ordinary skill in the art. For example, size exclusion chromatography media (e.g., CL-4B) can be used to determine the relative molecular size distribution of the conjugate. Size exclusion chromatography (SEC) can be used in gravity fed columns to profile the molecular size distribution of conjugates. Large molecules excluded from the pores in the media elute more quickly than small molecules. Fraction collectors can be used to collect the column eluate. The fractions can be tested colorimetrically by saccharide assay or by other methods. For the determination of Kd, columns can be calibrated to establish the fraction at which molecules are fully excluded (Vo), (Kd=0), and the fraction representing the maximum retention (Vi), (Kd=1). The fraction at which a specified sample attribute is reached (Ve), is related to Kd by the expression, Kd = (Ve - Vo)/ ( - Vo).
In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of glycoconjugates have a Kd below or equal to 0.3, for example, as determined using a CL-4B column.
Glycoconjugate yield
In some embodiments, after purification, the yield of glycoconjugate produced by carrying out a conjugation reaction using activated saccharide and carrier protein as described herein can be determined and expressed as percentage yield. The yield of glycoconjugate can be determined using routine methods known to those of skill in the art. In some embodiments, the yield of glycoconjugate produced by carrying out a conjugation reaction using activated saccharide and carrier protein as described herein is at least or about 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 85%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or some other value between or range encompassing any of the foregoing specifically enumerated values, or some other value or range.
Immunogenic compositions
In some embodiments, the disclosure provides an immunogenic composition comprising at least one glycoconjugate produced according to the methods described herein. Among other applications, such immunogenic compositions can be used in the prevention and/or treatment of diseases or disorders in a subject caused by microorganisms, such as bacteria, which produce the same or similar type of saccharide which is present in the glycoconjugate in the immunogenic composition.
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates of at least two distinct types, the first comprising a first saccharide and the second comprising a second saccharide which is immunologically distinct from the first saccharide. In some embodiments, the carrier protein of the first and at least second glycoconjugates is the same type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the carrier protein of the first and at least second glycoconjugates are different types of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein). In some embodiments, the saccharides of the first and at least second glycoconjugates are from distinct serotypes of the same species of bacteria, whereas in other embodiments, the saccharides of the first and at least second glycoconjugates are from distinct species of bacteria. In some embodiments, the saccharides of the first and at least second glycoconjugates are from distinct serotypes of S. pneumoniae bacteria.
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 20 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 20 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 21 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 21 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 22 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 22 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein). In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 23 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 23 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 24 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 24 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 25 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 25 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 26 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 26 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 27 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 27 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 28 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 28 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 29 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 29 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 30 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 30 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 31 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 31 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 32 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 32 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 33 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 33 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 34 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 34 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 35 or more serotypes of S. pneumoniae. In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising glycoconjugates between carrier protein and bacterial capsular saccharides from 35 or more serotypes of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3 and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11 A, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 15A and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 3, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11 A, 12F, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein). In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 23B and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 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, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 24F and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 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, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 35B and at least a second glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 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, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a first glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3, a second conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F, and at least a third glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11 A, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a first glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3, a second conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F, a third conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 15A, and at least a fourth glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23B, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a first glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3, a second conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F, a third conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 15A, a fourth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 23B, and at least a fifth glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23F, 24B, 24F, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a first glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3, a second conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F, a third conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 15A, a fourth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 23B, a fifth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 24F, and at least a sixth glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23F, 24B, 27, 29, 31 , 33B, 33F, 34, 35B, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
In some embodiments, the immunogenic composition is a multivalent immunogenic composition comprising a first glycoconjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 3, a second conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 12F, a third conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 15A, a fourth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 23B, a fifth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 24F, a sixth conjugate between CRM197 and bacterial capsular saccharide from S. pneumoniae serotype 35B, and at least a seventh glycoconjugate between carrier protein and a bacterial capsular saccharide from a distinct serotype of S. pneumoniae selected from the group consisting of 1 , 2, 4, 5, 6A, 6B, 6C, 7C, 7F, 8, 9V, 9N, 10A, 10B, 11A, 14, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 20B, 21 , 22A, 22F, 23A, 23F, 24B, 27, 29, 31 , 33B, 33F, 34, 35F, 38, 72 and 73, others being possible. In some embodiments, the glycoconjugates of the immunogenic composition comprise a single type of carrier protein (e.g., DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197), whereas in other embodiments, the glycoconjugates comprise more than one type of carrier protein (e.g., CRM197 and SCP, or TT, or yet a different type of carrier protein).
Dosages of immunogenic compositions
The amount of glycoconjugate(s) in each dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented.
The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on total saccharide for that conjugate (conjugated and non-conjugated). For example, a glycoconjugate with 20% free saccharide will have about 80 pg of conjugated saccharide and about 20 pg of nonconjugated saccharide in a 100 pg saccharide dose. The amount of glycoconjugate can vary depending upon the bacteria and bacteria serotype. The saccharide concentration can be determined by the uronic acid assay. The "immunogenic amount" of the different saccharide components in the immunogenic composition, may diverge and each may comprise about 0.5 pg, about 0.75 pg, about 1 pg, about 2 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, about 10 pg, about 15 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, or about 100 pg of any particular saccharide antigen.
Generally, each dose will comprise 0.1 pg to 100 pg of saccharide. In some embodiments each dose will comprise 0.1 pg to 100 pg of saccharide. In some embodiments each dose will comprise 0.5 pg to 20 pg. In some embodiments each dose will comprise 1.0 pg to 10 pg. In some other embodiments, each dose will comprise 2.0 pg to 5.0 pg of serotype 3 saccharide. Any whole number integer within any of the above ranges is contemplated as some embodiments of the disclosure.
In some embodiments, each dose will comprise about 0.5 pg of saccharide. In some embodiments, each dose will comprise about 0.55 pg of saccharide. In some embodiments, each dose will comprise about 0.75 pg of saccharide. In some embodiments, each dose will comprise about 1.0 pg of saccharide. In some embodiments, each dose will comprise about 1.1 pg of saccharide. In some embodiments, each dose will comprise about 1.5 pg of saccharide. In some embodiments, each dose will comprise about 2.0 pg of saccharide. In some embodiments, each dose will comprise about 2.2 pg of saccharide. In some embodiments, each dose will comprise about 2.5 pg of saccharide. In some embodiments, each dose will comprise about 3.0 pg of saccharide. In some embodiments, each dose will comprise about 3.5 pg of saccharide. In some embodiments, each dose will comprise about 4.0 pg of saccharide. In some embodiments, each dose will comprise about 4.4 pg of saccharide. In some embodiments, each dose will comprise about 5.0 pg of saccharide. In some embodiments, each dose will comprise about 5.5 pg of saccharide. In some embodiments, each dose will comprise about 6.0 pg of saccharide.
Generally, each dose will comprise 0.1 pg to 100 pg of saccharide for a given bacteria or serotype. In some embodiments each dose will comprise 0.1 pg to 100 pg of saccharide for a given bacteria or serotype. In some embodiments each dose will comprise 0.5 pg to 20 pg. In some embodiments each dose will comprise 1.0 pg to 10 pg. In other embodiments, each dose will comprise 2.0 pg to 5.0 pg of accharide for a given bacteria or serotype. Any whole number integer within any of the above ranges is contemplated as some embodiments of the disclosure.
In some embodiments, each dose will comprise about 0.5 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 0.55 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 0.75 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 1.0 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 1.1 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 1.5 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 2.0 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 2.2 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 2.5 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 3.0 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 3.5 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 4.0 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 4.4 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 5.0 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 5.5 pg of saccharide for each particular glycoconjugate. In some embodiments, each dose will comprise about 6.0 pg of saccharide for each particular glycoconjugate.
Carrier amount
Generally, each dose will comprise 10 pg to 150 pg of carrier protein, particularly 15 pg to 100 pg of carrier protein, more particularly 25 pg to 75 pg of carrier protein, and even more particularly 40 pg to 60 pg of carrier protein. In some embodiments, the carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197, or some other carrier protein.
In some embodiments, each dose will comprise about 10 pg, about 15 pg, about 20 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg. about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg or about 75 pg of carrier protein.
In some embodiments, each dose will comprise about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 31 pg, about 32 pg, about 33 pg, about 34 pg, about 35 pg, about 36 pg, about 37 pg, about 38 pg, about 39 pg, about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg or about 75 pg of carrier protein.
In some embodiments, each dose will comprise about 30 pg of carrier protein. In some embodiments, each dose will comprise about 31 pg of carrier protein. In some embodiments, each dose will comprise about 32 pg of carrier protein. In some embodiments, each dose will comprise about 33 pg of carrier protein. In some embodiments, each dose will comprise about 34 g of carrier protein. In some embodiments, each dose will comprise about 45 pg of carrier protein.
In some embodiments, each dose will comprise about 40 pg of carrier protein. In some embodiments, each dose will comprise about 41 pg of carrier protein. In some embodiments, each dose will comprise about 42 pg of carrier protein. In some embodiments, each dose will comprise about 43 pg of carrier protein. In some embodiments, each dose will comprise about 44 pg of carrier protein. In some embodiments, each dose will comprise about 45 pg of carrier protein.
In some embodiments, each dose will comprise about 48 pg of carrier protein. In some embodiments, each dose will comprise about 49 pg of carrier protein. In some embodiments, each dose will comprise about 50 pg of carrier protein. In some embodiments, each dose will comprise about 51 pg of carrier protein. In some embodiments, each dose will comprise about 52 pg of carrier protein. In some embodiments, each dose will comprise about 53 pg of carrier protein.
In some embodiments, the carrier protein is DT, TT, fHBP, PD, SCP, or CRM protein, such as CRM197, or some other carrier protein.
Adjuvants
In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two or three adjuvants. In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise one adjuvant. In some embodiments, the immunogenic compositions disclosed herein may further comprise two adjuvants. The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. Antigens may act primarily as a delivery system, primarily as an immune modulator or have strong features of both.
Suitable adjuvants include those suitable for use in mammals, including humans. Examples of known suitable delivery-system type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.
In some embodiments, the immunogenic compositions disclosed herein comprise aluminum salts (alum) as adjuvant (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide). In some embodiments, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as adjuvant. In some embodiments, the immunogenic compositions disclosed herein comprise aluminum phosphate as adjuvant.
Further exemplary adjuvants to enhance effectiveness of the immunogenic compositions as disclosed herein include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121 , and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (b) RIBI™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), for example, MPL + CWS (DETOX™); (2) saponin adjuvants, such as QS21 , STIMULON™ (Cambridge Bioscience, Worcester, MA), ABISCO® (Isconova, Sweden), or ISCOMATRIX® (Commonwealth Serum Laboratories, Australia), may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent (e.g., WO 00/07621); (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g., IL-1 , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g., WO 99/44636)), interferons (e.g., gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB-2220221 , EP0689454), optionally in the substantial absence of alum when used with pneumococcal saccharides (see, e.g., WO 00/56358); (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions (see, e.g., EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or a polyoxyethylene ester (see, e.g., WO 99/52549); (8) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g., WO 01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g., WO 01/21152); (9) a saponin and an immunostimulatory oligonucleotide (e.g., a CpG oligonucleotide) (e.g., WO 00/62800); (10) an immunostimulant and a particle of metal salt (see, e.g., WO 00/23105); (11) a saponin and an oil- in-water emulsion (e.g., WO 99/11241); (12) a saponin (e.g., QS21) + 3dMPL + IM2 (optionally + a sterol) (e.g., WO 98/57659); (13) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Muramyl peptides include N-acetyl-muramyl-L-threonyl- D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.
In some embodiments of the present invention, the immunogenic compositions as disclosed herein comprise a CpG Oligonucleotide as adjuvant. A CpG oligonucleotide as used herein refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and accordingly these terms are used interchangeably unless otherwise indicated. Immunostimulatory CpG oligodeoxynucleotides contain one or more immunostimulatory CpG motifs that are unmethylated cytosine-guanine dinucleotides, optionally within certain base contexts. The methylation status of the CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide which contains a 5' unmethylated cytosine linked by a phosphate bond to a 3' guanine, and which activates the immune system through binding to Toll-like receptor 9 (TLR-9). In another embodiment the immunostimulatory oligonucleotide may contain one or more methylated CpG dinucleotides, which will activate the immune system through TLR9 but not as strongly as if the CpG motif(s) was/were unmethylated. CpG immunostimulatory oligonucleotides may comprise one or more palindromes that in turn may encompass the CpG dinucleotide. CpG oligonucleotides have been described in a number of issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371 ; 6,239,116; and 6,339,068.
In some embodiments of the present invention, the immunogenic compositions as disclosed herein comprise any of the CpG Oligonucleotide described at page 3, line 22, to page 12, line 36, of WO 2010/125480.
Different classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as A, B, C and P class, and are described in greater detail at page 3, line 22, to page 12, line 36, of WO 2010/125480. Methods embrace the use of these different classes of CpG immunostimulatory oligonucleotides.
Formulations
The immunogenic compositions may be formulated in liquid form (i.e., solutions or suspensions) or in a lyophilized form. In some embodiments, the immunogenic composition is formulated in a liquid form. In some embodiments, the immunogenic composition is formulated in a lyophilized form. Liquid formulations may advantageously be administered directly from their packaged form and are thus ideal for injection without the need for reconstitution in aqueous medium as otherwise required for lyophilized compositions of the invention.
Formulation of the immunogenic composition of the present disclosure can be accomplished using art-recognized methods. For instance, the individual saccharides and/or conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
The present disclosure provides an immunogenic composition comprising any of combination of glycoconjugates disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.
In some embodiments, the immunogenic composition of the disclosure is in liquid form, for example, in aqueous liquid form.
Immunogenic compositions of the disclosure may comprise one or more of a buffer, a salt, a divalent cation, a non-ionic detergent, a cryoprotectant such as a sugar, and an anti-oxidant such as a free radical scavenger or chelating agent, or any multiple combinations thereof.
In some embodiments, the immunogenic compositions of the disclosure comprise a buffer. In some embodiments, said buffer has a pKa of about 3.5 to about 7.5. In some embodiments, the buffer is phosphate, succinate, histidine or citrate. In some embodiments, the buffer is succinate. In some embodiments, the buffer is histidine. In certain embodiments, the buffer is succinate at a final concentration of 1 mM to 10 mM. In one particular embodiment, the final concentration of the succinate buffer is about 5 mM.
In some embodiments, the immunogenic compositions of the disclosure comprise a salt. In some embodiments, the salt is selected from the groups consisting of magnesium chloride, potassium chloride, sodium chloride and a combination thereof. In one particular embodiment, the salt is sodium chloride. In one particular embodiment, the immunogenic compositions comprise sodium chloride at 150 mM.
In some embodiments, the immunogenic compositions of the disclosure comprise a surfactant. In some embodiments, the surfactant is selected from the group consisting of polysorbate 20 (TWEENTM20), polysorbate 40 (TWEENTM40), polysorbate 60 (TWEEN™60), polysorbate 65 (TWEEN™65), polysorbate 80 (TWEEN™80), polysorbate 85 (TWEEN™85), TRITON™ N-101 , TRITON™ X-100, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxy stearate (PEG-15, Solutol H 15), polyoxyethylene-35-ricinoleate (CREMOPHOR® EL), soy lecithin and a poloxamer.
In one particular embodiment, the surfactant is polysorbate 80. In some said embodiment, the final concentration of polysorbate 80 in the formulation is at least 0.0001 % to 10% polysorbate 80 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.001 % to 1% polysorbate 80 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 80 in the formulation is at least 0.01% to 1% polysorbate 80 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 80 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.02% polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.01 % polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.03% polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.04% polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 0.05% polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 80 in the formulation is 1% polysorbate 80 (w/w).
In one particular embodiment, the surfactant is polysorbate 20. In some said embodiment, the final concentration of polysorbate 20 in the formulation is at least 0.0001 % to 10% polysorbate 20 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.001 % to 1% polysorbate 20 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 20 in the formulation is at least 0.01% to 1% polysorbate 20 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 20 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 20 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.02% polysorbate 20 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.01 % polysorbate 20 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.03% polysorbate 20 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.04% polysorbate 80 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 0.05% polysorbate 20 (w/w). In another embodiment, the final concentration of the polysorbate 20 in the formulation is 1% polysorbate 20 (w/w).
In one particular embodiment, the surfactant is polysorbate 40. In some said embodiment, the final concentration of polysorbate 40 in the formulation is at least 0.0001 % to 10% polysorbate 40 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.001 % to 1% polysorbate 40 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 40 in the formulation is at least 0.01 % to 1% polysorbate 40 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 40 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 40 (w/w). In another embodiment, the final concentration of the polysorbate 40 in the formulation is 1% polysorbate 40 (w/w).
In one particular embodiment, the surfactant is polysorbate 60. In some said embodiment, the final concentration of polysorbate 60 in the formulation is at least 0.0001 % to 10% polysorbate 60 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.001 % to 1% polysorbate 60 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 60 in the formulation is at least 0.01% to 1% polysorbate 60 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 60 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 60 (w/w). In another embodiment, the final concentration of the polysorbate 60 in the formulation is 1% polysorbate 60 (w/w).
In one particular embodiment, the surfactant is polysorbate 65. In some said embodiment, the final concentration of polysorbate 65 in the formulation is at least 0.0001 % to 10% polysorbate 65 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.001 % to 1% polysorbate 65 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 65 in the formulation is at least 0.01% to 1% polysorbate 65 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 65 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 65 (w/w). In another embodiment, the final concentration of the polysorbate 65 in the formulation is 1% polysorbate 65 (w/w).
In one particular embodiment, the surfactant is polysorbate 85. In some said embodiment, the final concentration of polysorbate 85 in the formulation is at least 0.0001 % to 10% polysorbate 85 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.001 % to 1% polysorbate 85 weight to weight (w/w). In some said embodiments, the final concentration of polysorbate 85 in the formulation is at least 0.01% to 1% polysorbate 85 weight to weight (w/w). In other embodiments, the final concentration of polysorbate 85 in the formulation is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1 % polysorbate 85 (w/w). In another embodiment, the final concentration of the polysorbate 85 in the formulation is 1% polysorbate 85 (w/w).
In certain embodiments, the immunogenic composition of the disclosure has a pH of 5.5 to 7.5, for example, a pH of 5.6 to 7.0, or a pH of 5.8 to 6.0.
In some embodiments, the present disclosure provides a container filled with any of the immunogenic compositions disclosed herein. In some embodiments, the container is selected from the group consisting of a vial, a syringe, a flask, a fermentor, a bioreactor, a bag, a jar, an ampoule, a cartridge and a disposable pen. In certain embodiments, the container is siliconized.
In some embodiments, the container of the present disclosure is made of glass, metals (e.g., steel, stainless steel, aluminum, etc.) and/or polymers (e.g., thermoplastics, elastomers, thermoplastic-elastomers). In some embodiments, the container of the present disclosure is made of glass.
In some embodiments, the present disclosure provides a syringe filled with any of the immunogenic compositions disclosed herein. In certain embodiments, the syringe is siliconized and/or is made of glass.
A typical dose of the immunogenic composition for injection has a volume of 0.1 mL to 2 mL. In some embodiments, the immunogenic composition for injection has a volume of 0.2 mL to 1 mL, for example, a volume of about 0.5 mL.
Uses of glycoconjugate and immunogenic composition
The glycoconjugates disclosed herein may be use as antigens. For example, they may be part of a vaccine.
Therefore, in some embodiments, the immunogenic compositions are for use as a medicament.
In some embodiments, the immunogenic compositions are for use as a vaccine.
Therefore, in some embodiments, the immunogenic compositions described herein are for use in generating an immune response in a subject. In one aspect, the subject is a mammal, such as a human, non-human primate, cat, sheep, pig, horse, bovine or dog. In one aspect, the subject is a human.
The immunogenic compositions described herein may be used in therapeutic or prophylactic methods for preventing, treating or ameliorating a bacterial infection, disease or condition in a subject. Thus, in one aspect, the disclosure provides a method of preventing, treating or ameliorating an infection, disease or condition associated with a bacterial infection in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the disclosure. The immunogenic composition of the present disclosure can be used to protect or treat a human susceptible to a bacterial infection, by means of administering the immunogenic composition via a systemic or mucosal route. In some embodiments, the immunogenic composition is administered by intramuscular, intraperitoneal, intradermal or subcutaneous routes. In some embodiments, the immunogenic composition is administered by intramuscular, intraperitoneal, intradermal or subcutaneous injection. In some embodiments, the immunogenic composition is administered by intramuscular or subcutaneous injection. In some embodiments, the immunogenic composition is administered by intramuscular injection. In some embodiments, the immunogenic composition is administered by subcutaneous injection.
As used herein, a “prophylactically effective amount,” means an amount of a glycoconjugate, or an immunogenic composition comprising a glycoconjugate, which is effective, when administered to a subject, to prevent, at least in part, any symptom or sign of a disease or disorder caused by infection with an organism having a capsular saccharide of the same type as that which is present in the glycoconjugate or immunogenic composition.
As used herein, a “therapeutically effective amount,” means an amount of a glycoconjugate, or an immunogenic composition comprising a glycoconjugate, which is effective, when administered to a subject, to treat, at least in part, any symptom or sign of a disease or disorder caused by infection with an organism having a capsular saccharide of the same type as that which is present in the glycoconjugate or immunogenic composition.
As used herein, an “immunologically effective amount,” means an amount of a glycoconjugate, or an immunogenic composition comprising a glycoconjugate, which is effective, when administered to a subject, to elicit a cellular (including but not limited to T cell) and/or humoral (including but not limited to antibody) immune response, each as measured using standard assays known to those of ordinary skill in the art, against at least one capsular saccharide of the same type as that which is present in the glycoconjugate or immunogenic composition.
Subjects
As disclosed herein, the immunogenic compositions described herein may be used in various therapeutic or prophylactic methods for preventing, treating or ameliorating a bacterial infection, disease or condition in a subject.
In some embodiments, said subject is a human, including where such human subject is a newborn (i.e. , under three months of age), an infant (i.e. , from 3 months to one year of age) or a toddler (i.e., from one year to four years of age).
In some embodiments, the immunogenic compositions disclosed herein are for use as a vaccine.
In such embodiment, the subject to be vaccinated may be less than 1 year of age. For example, the subject to be vaccinated can be about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 months of age. In some embodiments, the subject to be vaccinated is about 2, about 4 or about 6 months of age. In another embodiment, the subject to be vaccinated is less than 2 years of age. For example, the subject to be vaccinated can be about 12 to about 15 months of age. In some cases, as little as one dose of the immunogenic composition according to the invention is needed, but under some circumstances, a second, third or fourth dose may be given (see section 8 below).
In some embodiments of the present invention, the subject to be vaccinated is a human adult 50 years of age or older, such as a human adult 55 years of age or older. In some embodiments, the subject to be vaccinated is a human adult 65 years of age or older, 70 years of age or older, 75 years of age or older or 80 years of age or older.
In some embodiments the subject to be vaccinated is an immunocompromised individual, in particular a human. An immunocompromised individual is generally defined as a person who exhibits an attenuated or reduced ability to mount a normal humoral or cellular defense to challenge by infectious agents.
In some embodiments of the present invention, the immunocompromised subject to be vaccinated suffers from a disease or condition that impairs the immune system and results in an antibody response that is insufficient to protect against or treat pneumococcal disease.
In some embodiments, said disease is a primary immunodeficiency disorder. In some embodiments, said primary immunodeficiency disorder is selected from the group consisting of: combined T- and B-cell immunodeficiencies, antibody deficiencies, well-defined syndromes, immune dysregulation diseases, phagocyte disorders, innate immunity deficiencies, autoinflammatory disorders, and complement deficiencies.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated suffers from a disease selected from the groups consisting of: HIV-infection, acquired immunodeficiency syndrome (AIDS), cancer, chronic heart or lung disorders, congestive heart failure, diabetes mellitus, chronic liver disease, alcoholism, cirrhosis, spinal fluid leaks, cardiomyopathy, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), spleen dysfunction (such as sickle cell disease), lack of spleen function (asplenia), blood malignancy, leukemia, multiple myeloma, Hodgkin’s disease, lymphoma, kidney failure, nephrotic syndrome and asthma.
In some embodiments of the present invention, the immunocompromised subject to be vaccinated suffers from malnutrition.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated is taking a drug or treatment that lowers the body’s resistance to infection.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated is a smoker.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated has a white blood cell count (WBC, or leukocyte count) below 5 x 109 cells per liter, or below 4 x 109 cells per liter, or below 3 x 109 cells per liter, or below 2 x 109 cells per liter, or below 1 x 109 cells per liter, or below 0.5 x 109 cells per liter, or below 0.3 x 109 cells per liter, or below 0.1 x 109 cells per liter. WBC can be measured as part of the CBC (complete blood count). White blood cells are distinct from erythrocytes. The normal range for the white blood cell count is usually between 4,300 and 10,800 cells per cubic millimeter of blood. This can also be referred to as the leukocyte count and can be expressed in international units as 4.3 - 10.8 x 109 cells per liter.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated suffers from neutropenia. In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated has a neutrophil count below 2 x 109 cells per liter, or below 1 x 109 cells per liter, or below 0.5 x 109 cells per liter, or below 0.1 x 109 cells per liter, or below 0.05 x 109 cells per liter.
A low white blood cell count or “neutropenia” is a condition characterized by abnormally low levels of neutrophils in the circulating blood. Neutrophils are a specific kind of white blood cell that help to prevent and fight infections. The most common reason that cancer patients experience neutropenia is as a side effect of chemotherapy. Chemotherapy-induced neutropenia increases a patient’s risk of infection and disrupts cancer treatment.
In a particular embodiment of the present invention, the immunocompromised subject to be vaccinated has a CD4+ cell count below 500/mm3, or CD4+ cell count below 300/mm3, or CD4+ cell count below 200/mm3, CD4+ cell count below 100/mm3, CD4+ cell count below 75/mm3, or CD4+ cell count below 50/mm3.
CD4 cell tests are normally reported as the number of cells in mm3. Normal CD4 counts are between 500 and 1 ,600, and CD8 counts are between 375 and 1 ,100. CD4 counts drop dramatically in people with HIV.
In some embodiments of the invention, any of the immunocompromised subjects disclosed herein is a human male. In some embodiments of the invention, any of the immunocompromised subjects disclosed herein is a human female.
Treatment regimens
In some cases, as little as one dose of the immunogenic composition according to the invention is needed, but under some circumstances, such as conditions of greater immune deficiency, a second, third or fourth dose may be given. Following an initial vaccination, subjects can receive one or several booster immunizations adequately spaced.
In some embodiments, the schedule of vaccination of the immunogenic composition according to the invention is a single dose. In a particular embodiment, said single dose schedule is for healthy persons being at least 2 years of age.
In some embodiments, the schedule of vaccination of the immunogenic composition according to the invention is a multiple dose schedule. In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 1 month to about 2 months. In a particular embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 1 month, or a series of 2 doses separated by an interval of about 2 months.
In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month to about 2 months. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month, or a series of 3 doses separated by an interval of about 2 months.
In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month to about 2 months followed by a fourth dose about 10 months to about 13 months after the first dose. In another embodiment, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month followed by a fourth dose about 10 months to about 13 months after the first dose, or a series of 3 doses separated by an interval of about 2 months followed by a fourth dose about 10 months to about 13 months after the first dose.
In some embodiments, the multiple dose schedule consists of at least one dose (e.g., 1, 2 or 3 doses) in the first year of age followed by at least one toddler dose.
In some embodiments, the multiple dose schedule consists of a series of 2 or 3 doses separated by an interval of about 1 month to about 2 months (for example 28-56 days between doses), starting at 2 months of age, and followed by a toddler dose at 12-18 months of age. In some embodiments, said multiple dose schedule consists of a series of 3 doses separated by an interval of about 1 month to about 2 months (for example 28-56 days between doses), starting at 2 months of age, and followed by a toddler dose at 12-15 months of age. In another embodiment, said multiple dose schedule consists of a series of 2 doses separated by an interval of about 2 months, starting at 2 months of age, and followed by a toddler dose at 12-18 months of age.
In some embodiments, the multiple dose schedule consists of a 4-dose series of vaccine at 2, 4, 6, and 12-15 months of age.
In some embodiments, a prime dose is given at day 0 and one or more boosts are given at intervals that range from about 2 to about 24 weeks, for example in a dosing interval of 4-8 weeks.
In some embodiments, a prime dose is given at day 0 and a boost is given about 3 months later.
Other objects, features and advantages of the present invention will be apparent from the foregoing detailed description. It should be understood, however, that the detailed description and the specific examples that follow, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes, modifications and equivalents within the spirit and scope will be apparent from the detailed description and examples to those of ordinary skill in the art and fall within the scope of the appended claims. Unless otherwise indicated, use of the term “or” in reference to one or more members of a set of embodiments is equivalent in meaning to “and/or,” and does not require that they be mutually exclusive of each other. Unless otherwise indicated, a plurality of expressly recited numeric ranges also describes a range the lower bound of which is derived from the lower or upper bound of any one of the expressly recited ranges, and the upper bound of which is derived from the lower or upper bound of any other of the expressly recited ranges. Thus, for example, the series of expressly recited ranges “10-20, 20-30, 30-40, 40-50, 100-150, 200-250, 275-300,” also describes the ranges 10-50, 50-100, 100-200, and 150-250, among many others. Unless otherwise indicated, use of the term “about” before a series of numerical values or ranges is intended to modify not only the value or range appearing immediately after it but also each and every value or range appearing thereafter in the same series. Thus, for example, the phrase “about 1 , 2, or 3,” is equivalent to “about 1 , about 2, or about 3.”
All publications and references, including but not limited to articles, abstracts, patents, patent applications (whether published or unpublished), biological sequences (including, but not limited to those identified by specific database reference numbers or sequence listing numbers from patents or published patent applications) cited herein are hereby incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, reference, or biological sequence was specifically and individually indicated to be so incorporated by reference.
Any patent application to which this application claims priority directly or indirectly is incorporated herein by reference in its entirety for all purposes to the same extent as if each individual patent application was specifically and individually indicated to be so incorporated by reference.
Unless otherwise indicated, the examples below describe experiments that were or are performed using standard techniques well known and routine to those of ordinary skill in the art. The examples are illustrative, but do not limit the invention.
EXAMPLES
Example 1 : Development of a novel CRM compounding formulation and its use to prepare Pn15A glycoconjugates
The inventors discovered that producing glycoconjugates with capsular saccharides from certain Streptococcus pneumoniae serotypes using reductive amination in DMSO could not be carried out efficiently at the concentrations of saccharide (about 1 g/L) typically used in prevailing methods, for example, those used to produce glycoconjugates in PCV13 vaccine. Although it was hypothesized that increasing the concentration of saccharide in the reaction could improve efficiency and conjugate quality, the inventors identified a new problem with that approach. More specifically, increasing the saccharide concentration required increasing the CRM197 carrier protein concentration in the reaction as well, but its solubility after lyophilization and reconstitution in DMSO from the existing compounding formulation was limited to 3 g/L or less, which was too low. Thus, to conjugate certain saccharide serotypes more efficiently first required increasing CRM197 solubility in DMSO.
Before lyophilization, the existing CRM197 compounding formulation contained 15% sucrose (w/v) and 72 mM NaCI in 15 mM sodium phosphate buffer (pH 7.4). In the typical method, lyophilization and reconstituting this formulation in DMSO accommodated a CRM197 protein concentration of about 2.0 to 2.5 g/L, which was sufficient to carry out reductive amination using saccharide serotypes found in earlier generations of PCV vaccines. It was hypothesized however, that ionic composition and/or relatively high sucrose concentration (25x mass relative to the amount of CRM197 protein present) might interfere with CRM197 protein solubility in DMSO. The effect of changing these variables on solubility was therefore investigated.
The existing CRM197 compounding formulation was buffer exchanged with 5 mM potassium phosphate buffer (pH 7.4), thus replacing the NaCI, sodium phosphate buffer, and sucrose. Briefly, the calculated amount of CRM197 protein in the old formulation was transferred to the LIF/DF vessel. The diafiltration was performed using 20-fold diavolume of 5mM potassium phosphate, pH 7.4. The target settings and ranges for diafiltration includes a membrane challenge of 1 - 10 grams of protein per ft2 of membrane area, transmembrane pressure (TMP) of 5 to 30 psi and target retentate concentration of 6.0 ± 1.0 g/L.
The new potassium-based formulation was then supplemented with increasing amounts of sucrose. Briefly, the buffer exchanged CRM197 protein formulation was compounded with aqueous 50% sucrose solution to ratios of 2.5, 5.0, 7.5 or 10.0 grams sucrose per gram CRM197 protein, and final CRM197 protein concentration adjusted to 6.0 ± 1.0 g/L using 5 mM potassium phosphate buffer. A fifth formulation was prepared by adding 2.5x sucrose and KCI to a final concentration of 12 mM to increase the potassium ion concentration. The new CRM197 formulations were shell frozen in an ethanol bath at -25 to -90 °C, lyophilized and stored up to 12 months at -80 ± 10 °C.
After reconstitution, conjugating CRM197 with activated S. pneumoniae serotype 15 saccharide by reductive amination in DMSO was compared using the original version of the CRM197 protein compounding formulation and the new versions containing potassium. Briefly, lyophilized activated Pn15A saccharide was reconstituted to 8 g/L in anhydrous DMSO. CRM197 protein, which had been separately lyophilized in the new compounding formulations, was reconstituted in the same amount of anhydrous DMSO to a concentration of 8.0 g/L. The reconstituted solutions were combined in a reactor, followed by mixing for 60 to 120 minutes at
23 ± 2 °C, during which the moisture level was monitored. The final saccharide concentration was 4.0 g/L and final CRM197 protein concentration was 4.0 g/L. Conjugation was initiated by adding 1.0 molar equivalents of sodium cyanoborohydride (NaCNBHs) and then proceeded for
24 ± 4 hrs. To terminate the conjugation reaction, 2 molar equivalents sodium borohydride (NaBH4) was added as a capping reagent, followed by further incubation for 3 hrs at 23 ± 2 °C. The conjugate solution was then diluted into 4 to 9x (by volume) chilled 5mM succinate/saline (pH 6.0), mixed for 10 minutes, filtered through a 5pm filter, and concentrated to 1 to 5 g/L using 100K MWCO regenerated cellulose membranes with diafiltration against 5mM succinate/saline (pH 6.0). Diafiltration conditions included a membrane challenge of 1 to 10 grams of saccharide per ft2 of membrane area, transmembrane pressure (TMP) of 5 to 20 psi, and target retentate concentration of 1 to 5 g/L. Following diafiltration, conjugate retentate was filtered through a 0.45/0.22pm filter into a collection bottle.
The original CRM197 compounding formulation, which allowed only low concentrations of activated Pn15A saccharide and carrier protein to be reacted, gave poor results. More specifically, with the original version, the reaction contained 1 g/L Pn15A saccharide (degree of oxidation 3.5, Mw 106 kDa), 1 to 1.25 g/L CRM197 protein, 5 to 5.6% sucrose and 12 mM NaCI. Conjugation proceeded slowly and produced low product yields of less than 10% with high free saccharide of greater than 70%. By contrast, the new formulations permitted conjugation to proceed at higher concentrations of saccharide and CRM197 protein and produced much better results.
Table 1 shows the effect of increasing sucrose concentration in the CRM197 compounding formulation upon conjugation with Pn15A saccharide. Overall, the effect was modest, with trends of increasing conjugate molecular weight and free saccharide as sucrose concentration increased.
Table 1
Table 2 shows the effect of increasing the potassium ion concentration in the CRM197 compounding formulation upon conjugation with activated Pn15A saccharide. Adding KOI to a final concentration of 12 mM to the potassium phosphate-based compounding formulation increased conjugate Mw substantially at two different Pn15A saccharide degrees of oxidation. Because excessively high conjugate Mw can make 0.22pm filtration challenging, the inventors determined that it was not necessary to supplement the CRM197 compounding formulation with KCI. Taking into consideration filterability and other factors such as structural appearance and hygroscopicity of the lyophilized cake, a CRM197 compounding formulation containing about 6 g/L CRM 7 protein and 4.5% sucrose (equivalent to 7.5x protein (w/w)) in 5mM potassium phosphate buffer (pH7.4) was identified as being superior to the conventional formulation containing sodium phosphate by dramatically increasing CRM197 protein solubility upon reconstitution in DMSO, making it possible to carry out conjugation by reductive amination using higher activated saccharide concentrations with a concomitant improvement in yield and conjugate quality.
Table 2
Finally, as shown in Table 3, the new CRM197 compounding formulation could be used for preparing Pn15A glycoconjugates by reductive amination in DMSO at larger scales suitable for preclinical studies and and clinical trials.
Table 3
Example 2: Preparation of Pn23B and Pn35B glycoconjugates
As discussed in Example 1 , the inventors developed an improved CRM197 compounding formulation which dramatically increased CRM197 protein solubility upon reconstitution in DMSO, making it possible to carry out reductive amination conjugation using higher concentrations of activated Pn15A saccharide with a concomitant improvement in yield and conjugate quality. The inventors then investigated whether the new compounding formulation could be used to improve conjugation by reductive amination in DMSO using activated saccharides from S. pneumoniae serotypes 23B and 35B, which had also proved challenging to conjugate at the lower concentrations required when using the previous CRM197 compounding formulation.
Lyophilized activated Pn23B saccharide was reconstituted to 6 g/L in anhydrous dimethyl sulfoxide (DMSO). CRM197 protein, which had been separately lyophilized in the new compounding formulation containing 7.5x sucrose in 5mM potassium phosphate buffer (pH7.4), was reconstituted in the same amount of anhydrous DMSO to a concentration of 6.6 g/L. The reconstituted solutions were combined in a reactor, followed by mixing for 60 to 120 minutes at
23 ± 2 °C, during which the moisture level was monitored. The final saccharide concentration was 3.0 g/L and final CRM197 protein concentration was 3.3 g/L. Conjugation was initiated by adding 1.0 molar equivalents of sodium cyanoborohydride (NaCNBHs) and then proceeded for
24 ± 4 hrs. To terminate the conjugation reaction, 2 molar equivalents sodium borohydride (NaBhL) was added as a capping reagent, followed by further incubation for 3 hrs at 23 ± 2 °C. The conjugate solution was then diluted into 4 to 9x (by volume) chilled 5mM succinate/saline (pH 6.0), mixed for 10 minutes, filtered through a 5pm filter, and concentrated to 1 to 5 g/L using 100K MWCO regenerated cellulose membranes with diafiltration against 5mM succinate/saline (pH 6.0). Diafiltration conditions included a membrane challenge of 1 to 10 grams of saccharide per ft2 of membrane area, transmembrane pressure (TMP) of 5 to 20 psi, and target retentate concentration of 1 to 5 g/L. Following diafiltration, conjugate retentate was filtered through a 0.45/0.22pm filter into a collection bottle. Following analysis, results are shown in Table 4, below.
Table 4
Lyophilized activated Pn35B saccharide was reconstituted to 8 g/L in anhydrous dimethyl sulfoxide (DMSO). CRM197 protein, which had been separately lyophilized in the new compounding formulation containing 7.5x sucrose in 5mM potassium phosphate buffer (pH7.4), was reconstituted in the same amount of anhydrous DMSO to a concentration of 10.0 g/L. The reconstituted solutions were combined in a reactor, followed by mixing for 60 to 120 minutes at
23 ± 2 °C, during which the moisture level was monitored. The final saccharide concentration was 4.0 g/L and final CRM197 protein concentration was 5.0 g/L. Conjugation was initiated by adding 1.0 molar equivalents of sodium cyanoborohydride (NaCNBHs) and then proceeded for
24 ± 4 hrs. To terminate the conjugation reaction, 2 molar equivalents sodium borohydride (NaBH4) was added as a capping reagent, followed by further incubation for 3 hrs at 23 ± 2 °C. Conjugates were then processed as above. Following analysis, results are shown in Table 4, below. Following analysis, results are shown in Table 5, below.
Table 5
Example 3: Preparation of Pn12F and Pn3 glycoconjugates
In attempting to prepare glycoconjugates with activated Pn12F saccharide, the inventors determined that it was possible to conjugate NalC -activated Pn12F saccharide with CRM197 protein using reductive amination in aqueous solvent, but the resulting conjugates had poor stability. Switching to reductive amination in DMSO was not feasible, however, because compounding the saccharide with lyophilized CRM197 protein reconstituted with DMSO from the conventional compounding formulation produced a precipitate before addition of NaCNBHs. Similarly, preparing conjugates with Pn3 saccharide by reductive amination in aqueous solvent was found to be suboptimal due to low yields of <30% and the observation that both conjugate size and immunogenicity decreased in cold storage. Solubility problems, however, prevented use of DMSO as a solvent. Specifically, the sodium salt of the negatively charged Pn3 saccharide is not soluble in DMSO at all, and both the potassium salt and acid forms of Pn3 saccharide are somewhat soluble in DMSO, but precipitate when compounded with CRM197 reconstituted with DMSO from the conventional compounding formulation containing sodium phosphate buffer. The inventors therefore investigated whether the new compounding formulation might be used to prepare glycoconjugates with activated saccharides from S. pneumoniae serotypes 12F and 3 by reductive amination in DMSO.
Lyophilized activated Pn12F saccharide was reconstituted to 2 g/L in anhydrous dimethyl sulfoxide (DMSO). CRM197 protein, which had been separately lyophilized in the new compounding formulation containing 7.5x sucrose in 5mM potassium phosphate buffer (pH7.4), was reconstituted in the same amount of anhydrous DMSO to a concentration of 2.5 g/L. The reconstituted solutions were combined in a reactor, followed by mixing for 60 to 120 minutes at
23 ± 2 °C, during which the moisture level was monitored. The final saccharide concentration was 1.0 g/L and final CRM197 protein concentration was 1.25 g/L. Conjugation was initiated by adding 1.0 molar equivalents of sodium cyanoborohydride (NaCNBHs) and then proceeded for
24 ± 4 hrs. To terminate the conjugation reaction, 2 molar equivalents sodium borohydride (NaBH4) was added as a capping reagent, followed by further incubation for 3 hrs at 23 ± 2 °C. Conjugates were then processed as above. Following analysis, results are shown in Table 6, below.
Table 6
Lyophilized activated Pn3 saccharide was reconstituted to 2 g/L in anhydrous dimethyl sulfoxide (DMSO). CRM197 protein, which had been separately lyophilized in the new compounding formulation containing 7.5x sucrose in 5mM potassium phosphate buffer (pH7.4), was reconstituted in the same amount of anhydrous DMSO to a concentration of 2.0 g/L. The reconstituted solutions were combined in a reactor, followed by mixing for 60 to 120 minutes at
23 ± 2 °C, during which the moisture level was monitored. The final saccharide concentration was 1.0 g/L and final CRM197 protein concentration was 1.0 g/L. Conjugation was initiated by adding 1.0 molar equivalents of sodium cyanoborohydride (NaCNBHs) and then proceeded for
24 ± 4 hrs. To terminate the conjugation reaction, 2 molar equivalents sodium borohydride (NaBhL) was added as a capping reagent, followed by further incubation for 3 hrs at 23 ± 2 °C. Conjugates were then processed as above. Following analysis, results are shown in Table 7, below.
Table 7
As can be seen from the results, use of reductive amination in DMSO to conjugate CRM197 protein and Pn12F and Pn3 saccharides produced high yields of both types of glycoconjugates in an acceptable range of molecular weights and with low amounts of free saccharide. Thus, use of the new CRM197 compounding formulation in potassium phosphate is an improvement over standard methods of making these conjugates by reductive amination in acqueous solvent, which was a compromise due to limitations associated with the inability to reconstitute CRM197 protein in DMSO when lyophilized in a compounding formulation containing sodium phosphate buffer. For Pn12F, reductive amination in DMSO with CRM197 reconstituted from the potassium phosphate compounding formulation permits use of NalO4 to activate the saccharide in place of TEMP/NCS (which was often associated with over-oxidation of about 15%), and offers the advantages of shorter conjugation time (24 hours versus 80 hours), streamlined ultrafiltration/diafiltration processing steps (into a 1 buffer system versus 3 buffer system), improved yield (>80% compared to about =50%), as well as increased conjugate homogeneity and stability. Similarly, for Pn3, reductive amination in DMSO with CRM197 reconstituted from the potassium phosphate compounding formulation permits use of NalC to activate the saccharide in place of HsIOe/MgCh, which results in a higher degree of oxidation (DO15 for NaIC versus DO5 for HsIOe/MgCh), and offers the advantages of shorter conjugation time (24 hours versus 48 hours), streamlined ultrafiltration/diafiltration processing steps (into a 1 buffer system versus 3 buffer system), improved yield (>75% compared to <30%), and increased conjugate homogeneity.

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 than
2 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 than
3 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.
PCT/IB2025/053995 2024-04-19 2025-04-16 Improved methods for producing glycoconjugates by reductive amination in aprotic solvent Pending WO2025219904A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463636217P 2024-04-19 2024-04-19
US63/636,217 2024-04-19

Publications (1)

Publication Number Publication Date
WO2025219904A1 true WO2025219904A1 (en) 2025-10-23

Family

ID=95656395

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2025/053995 Pending WO2025219904A1 (en) 2024-04-19 2025-04-16 Improved methods for producing glycoconjugates by reductive amination in aprotic solvent

Country Status (1)

Country Link
WO (1) WO2025219904A1 (en)

Citations (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4709017A (en) 1985-06-07 1987-11-24 President And Fellows Of Harvard College Modified toxic vaccines
GB2220221A (en) 1988-07-02 1990-01-04 Bkl Extrusions Ltd Glazing bead
EP0372501A2 (en) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Synthetic antigens, method for their preparation and their use
EP0378881A1 (en) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Synthetic peptides and their use as universal carriers for the preparation of immunogenic conjugates suitable for the development of synthetic vaccines
US4950740A (en) 1987-03-17 1990-08-21 Cetus Corporation Recombinant diphtheria vaccines
WO1991001146A1 (en) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine and hormone carriers for conjugate vaccines
EP0427347A1 (en) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Synthetic peptides useful as universal carriers for the preparation of immunogenic conjugates and their use in the development of synthetic vaccines
EP0471177A2 (en) 1990-08-13 1992-02-19 American Cyanamid Company Filamentous hemagglutinin of bordetella pertussis as a carrier molecule for conjugate vaccines
WO1993017712A3 (en) 1992-03-06 1993-11-11 Sclavo Biocine Spa Conjugates formed from heat shock proteins and oligo- or polysaccharides
WO1994003208A1 (en) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugates of poorly immunogenic antigens and synthetic peptide carriers and vaccines comprising them
EP0594610A1 (en) 1990-05-31 1994-05-04 Arne Forsgren PROTEIN D - AN IgD-BINDING PROTEIN OF HAEMOPHILUS INFLUENZAE
EP0689454A1 (en) 1993-03-23 1996-01-03 Smithkline Beecham Biolog Vaccine compositions containing 3-o deacylated monophosphoryl lipid a
EP0735898A1 (en) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines
EP0761231A1 (en) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccine composition containing adjuvants
US5614382A (en) 1993-03-05 1997-03-25 American Cyanamid Company Plasmid for production of CRM protein and diphtheria toxin
WO1997026008A1 (en) 1996-01-22 1997-07-24 Regents Of The University Of Minnesota STREPTOCOCCAL C5a PEPTIDASE VACCINE
EP0835318A2 (en) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines against hepatitis c
US5843711A (en) 1992-05-06 1998-12-01 The Regents Of The University Of California Diphtheria toxin receptor-binding region
WO1998057659A1 (en) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Adjuvant compositions for vaccines
WO1999011241A1 (en) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Oil in water emulsions containing saponins
WO1998058668A3 (en) 1997-06-20 1999-04-15 Microbiological Res Authority Bordetella pertussis antigens as carriers in vaccinating conjugates and oral vaccines comprising bordetella pertussis fimbriae
US5917017A (en) 1994-06-08 1999-06-29 President And Fellows Of Harvard College Diphtheria toxin vaccines bearing a mutated R domain
WO1999044636A2 (en) 1998-03-05 1999-09-10 The Medical College Of Ohio Il-12 enhancement of immune responses to t-independent antigens
WO1999052549A1 (en) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Adjuvant compositions
WO2000007621A2 (en) 1998-08-05 2000-02-17 Smithkline Beecham Biologicals S.A. Vaccine comprising an iscom consisting of sterol and saponin which is free of additional detergent
WO2000023105A2 (en) 1998-10-16 2000-04-27 Smithkline Beecham Biologicals S.A. Adjuvant systems and vaccines
WO2000034487A1 (en) 1998-12-07 2000-06-15 Regents Of The University Of Minnesota STREPTOCOCCAL C5a PEPTIDASE VACCINE
WO2000056358A2 (en) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccine against streptococcus pneumoniae capsular polysaccharides
WO2000062800A2 (en) 1999-04-19 2000-10-26 Smithkline Beecham Biologicals Sa Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide
WO2000039299A3 (en) 1998-12-23 2000-11-02 Iaf Biochem Int Streptococcus antigens
WO2000037105A3 (en) 1998-12-21 2000-11-09 Medimmune Inc Streptococcus pneumoniae proteins and immunogenic fragments for vaccines
WO2000061761A3 (en) 1999-04-09 2001-02-22 Techlab Inc Recombinant clostridium toxin a protein carrier for polysaccharide conjugate vaccines
US6194388B1 (en) 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2001021207A2 (en) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Use of combination of polyoxyethylene sorbitan ester and octoxynol as adjuvant and its use in vaccines
WO2001021152A1 (en) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Adjuvant comprising a polyxyethylene alkyl ether or ester and at least one nonionic surfactant
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO2001072337A1 (en) 2000-03-27 2001-10-04 Microbiological Research Authority Proteins for use as carriers in conjugate vaccines
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
WO2001098334A3 (en) 2000-06-20 2002-09-06 Shire Biochem Inc Streptococcus antigens
US6455673B1 (en) 1994-06-08 2002-09-24 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
WO2002091998A2 (en) 2001-05-11 2002-11-21 Aventis Pasteur, Inc. Novel meningitis conjugate vaccine
WO2003054007A2 (en) 2001-12-20 2003-07-03 Shire Biochem Inc. Streptococcus antigens
WO2004081515A2 (en) 2003-03-13 2004-09-23 Glaxosmithkline Biologicals S.A. Purification process for bacterial cytolysin
WO2004083251A2 (en) 2003-03-17 2004-09-30 Wyeth Holdings Corporation Mutant cholera holotoxin as an adjuvant and an antigen carrier protein
WO2006032499A1 (en) 2004-09-22 2006-03-30 Glaxosmithkline Biologicals S.A. Purification process for bacterial cytolysin
US20060228381A1 (en) 2005-04-08 2006-10-12 Wyeth Separation of contaminants from Streptococcus pneumoniae polysaccharide by pH manipulation
US20060228380A1 (en) 2005-04-08 2006-10-12 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070184072A1 (en) 2005-04-08 2007-08-09 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070184071A1 (en) 2005-04-08 2007-08-09 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070231340A1 (en) 2005-04-08 2007-10-04 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20080102498A1 (en) 2006-10-10 2008-05-01 Wyeth Methods for the separation of streptococcus pneumoniae type 3 polysaccharides
WO2008118752A2 (en) 2007-03-23 2008-10-02 Wyeth Shortened purification process for the production of capsular streptococcus pneumoniae polysaccharides
WO2009000826A1 (en) 2007-06-26 2008-12-31 Glaxosmithkline Biologicals S.A. Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates
WO2010125480A1 (en) 2009-04-30 2010-11-04 Coley Pharmaceutical Group, Inc. Pneumococcal vaccine and uses thereof
CN103495161A (en) 2013-10-08 2014-01-08 江苏康泰生物医学技术有限公司 Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture
US20200276316A1 (en) * 2017-02-24 2020-09-03 Merck Sharp & Dohme Corp. Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein
US20200306357A1 (en) * 2014-01-21 2020-10-01 Pfizer Inc. Immunogenic Compositions Comprising Conjugated Capsular Saccharide Antigens and Uses Thereof
US20210330777A1 (en) * 2017-02-24 2021-10-28 Merck Sharp & Dohme Corp. Enhancing immunogenicity of streptococcus pneumoniae polysaccharide-protein conjugates
JP7268125B2 (en) * 2015-07-21 2023-05-02 ファイザー・インク Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising same and uses thereof

Patent Citations (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4709017A (en) 1985-06-07 1987-11-24 President And Fellows Of Harvard College Modified toxic vaccines
US4950740A (en) 1987-03-17 1990-08-21 Cetus Corporation Recombinant diphtheria vaccines
GB2220221A (en) 1988-07-02 1990-01-04 Bkl Extrusions Ltd Glazing bead
EP0372501A2 (en) 1988-12-07 1990-06-13 BEHRINGWERKE Aktiengesellschaft Synthetic antigens, method for their preparation and their use
EP0378881A1 (en) 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Synthetic peptides and their use as universal carriers for the preparation of immunogenic conjugates suitable for the development of synthetic vaccines
WO1991001146A1 (en) 1989-07-14 1991-02-07 Praxis Biologics, Inc. Cytokine and hormone carriers for conjugate vaccines
EP0427347A1 (en) 1989-11-10 1991-05-15 ENIRICERCHE S.p.A. Synthetic peptides useful as universal carriers for the preparation of immunogenic conjugates and their use in the development of synthetic vaccines
EP0594610A1 (en) 1990-05-31 1994-05-04 Arne Forsgren PROTEIN D - AN IgD-BINDING PROTEIN OF HAEMOPHILUS INFLUENZAE
EP0471177A2 (en) 1990-08-13 1992-02-19 American Cyanamid Company Filamentous hemagglutinin of bordetella pertussis as a carrier molecule for conjugate vaccines
WO1993017712A3 (en) 1992-03-06 1993-11-11 Sclavo Biocine Spa Conjugates formed from heat shock proteins and oligo- or polysaccharides
US5843711A (en) 1992-05-06 1998-12-01 The Regents Of The University Of California Diphtheria toxin receptor-binding region
EP0761231A1 (en) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccine composition containing adjuvants
WO1994003208A1 (en) 1992-07-30 1994-02-17 Yeda Research And Development Company Ltd. Conjugates of poorly immunogenic antigens and synthetic peptide carriers and vaccines comprising them
US5614382A (en) 1993-03-05 1997-03-25 American Cyanamid Company Plasmid for production of CRM protein and diphtheria toxin
EP0689454A1 (en) 1993-03-23 1996-01-03 Smithkline Beecham Biolog Vaccine compositions containing 3-o deacylated monophosphoryl lipid a
EP0735898A1 (en) 1993-12-23 1996-10-09 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines
US5917017A (en) 1994-06-08 1999-06-29 President And Fellows Of Harvard College Diphtheria toxin vaccines bearing a mutated R domain
US6455673B1 (en) 1994-06-08 2002-09-24 President And Fellows Of Harvard College Multi-mutant diphtheria toxin vaccines
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6194388B1 (en) 1994-07-15 2001-02-27 The University Of Iowa Research Foundation Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
EP0835318A2 (en) 1995-06-29 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccines against hepatitis c
WO1997026008A1 (en) 1996-01-22 1997-07-24 Regents Of The University Of Minnesota STREPTOCOCCAL C5a PEPTIDASE VACCINE
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6339068B1 (en) 1997-05-20 2002-01-15 University Of Iowa Research Foundation Vectors and methods for immunization or therapeutic protocols
WO1998057659A1 (en) 1997-06-14 1998-12-23 Smithkline Beecham Biologicals S.A. Adjuvant compositions for vaccines
WO1998058668A3 (en) 1997-06-20 1999-04-15 Microbiological Res Authority Bordetella pertussis antigens as carriers in vaccinating conjugates and oral vaccines comprising bordetella pertussis fimbriae
WO1999011241A1 (en) 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Oil in water emulsions containing saponins
WO1999044636A2 (en) 1998-03-05 1999-09-10 The Medical College Of Ohio Il-12 enhancement of immune responses to t-independent antigens
US6218371B1 (en) 1998-04-03 2001-04-17 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
WO1999052549A1 (en) 1998-04-09 1999-10-21 Smithkline Beecham Biologicals S.A. Adjuvant compositions
WO2000007621A2 (en) 1998-08-05 2000-02-17 Smithkline Beecham Biologicals S.A. Vaccine comprising an iscom consisting of sterol and saponin which is free of additional detergent
WO2000023105A2 (en) 1998-10-16 2000-04-27 Smithkline Beecham Biologicals S.A. Adjuvant systems and vaccines
WO2000034487A1 (en) 1998-12-07 2000-06-15 Regents Of The University Of Minnesota STREPTOCOCCAL C5a PEPTIDASE VACCINE
WO2000037105A3 (en) 1998-12-21 2000-11-09 Medimmune Inc Streptococcus pneumoniae proteins and immunogenic fragments for vaccines
WO2000039299A3 (en) 1998-12-23 2000-11-02 Iaf Biochem Int Streptococcus antigens
WO2000056358A2 (en) 1999-03-19 2000-09-28 Smithkline Beecham Biologicals S.A. Vaccine against streptococcus pneumoniae capsular polysaccharides
WO2000061761A3 (en) 1999-04-09 2001-02-22 Techlab Inc Recombinant clostridium toxin a protein carrier for polysaccharide conjugate vaccines
WO2000062800A2 (en) 1999-04-19 2000-10-26 Smithkline Beecham Biologicals Sa Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide
WO2001021152A1 (en) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Adjuvant comprising a polyxyethylene alkyl ether or ester and at least one nonionic surfactant
WO2001021207A2 (en) 1999-09-24 2001-03-29 Smithkline Beecham Biologicals S.A. Use of combination of polyoxyethylene sorbitan ester and octoxynol as adjuvant and its use in vaccines
WO2001072337A1 (en) 2000-03-27 2001-10-04 Microbiological Research Authority Proteins for use as carriers in conjugate vaccines
WO2001098334A3 (en) 2000-06-20 2002-09-06 Shire Biochem Inc Streptococcus antigens
WO2002091998A2 (en) 2001-05-11 2002-11-21 Aventis Pasteur, Inc. Novel meningitis conjugate vaccine
WO2003054007A2 (en) 2001-12-20 2003-07-03 Shire Biochem Inc. Streptococcus antigens
WO2004081515A2 (en) 2003-03-13 2004-09-23 Glaxosmithkline Biologicals S.A. Purification process for bacterial cytolysin
WO2004083251A2 (en) 2003-03-17 2004-09-30 Wyeth Holdings Corporation Mutant cholera holotoxin as an adjuvant and an antigen carrier protein
WO2006032499A1 (en) 2004-09-22 2006-03-30 Glaxosmithkline Biologicals S.A. Purification process for bacterial cytolysin
US20060228381A1 (en) 2005-04-08 2006-10-12 Wyeth Separation of contaminants from Streptococcus pneumoniae polysaccharide by pH manipulation
US20060228380A1 (en) 2005-04-08 2006-10-12 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070184072A1 (en) 2005-04-08 2007-08-09 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070184071A1 (en) 2005-04-08 2007-08-09 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20070231340A1 (en) 2005-04-08 2007-10-04 Wyeth Multivalent pneumococcal polysaccharide-protein conjugate composition
US20080102498A1 (en) 2006-10-10 2008-05-01 Wyeth Methods for the separation of streptococcus pneumoniae type 3 polysaccharides
WO2008118752A2 (en) 2007-03-23 2008-10-02 Wyeth Shortened purification process for the production of capsular streptococcus pneumoniae polysaccharides
WO2009000826A1 (en) 2007-06-26 2008-12-31 Glaxosmithkline Biologicals S.A. Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates
WO2010125480A1 (en) 2009-04-30 2010-11-04 Coley Pharmaceutical Group, Inc. Pneumococcal vaccine and uses thereof
CN103495161A (en) 2013-10-08 2014-01-08 江苏康泰生物医学技术有限公司 Mixture of poly-pneumococcal capsular polysaccharide-protein conjugates and preparation method of mixture
US20200306357A1 (en) * 2014-01-21 2020-10-01 Pfizer Inc. Immunogenic Compositions Comprising Conjugated Capsular Saccharide Antigens and Uses Thereof
JP7268125B2 (en) * 2015-07-21 2023-05-02 ファイザー・インク Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising same and uses thereof
US20200276316A1 (en) * 2017-02-24 2020-09-03 Merck Sharp & Dohme Corp. Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein
US20210330777A1 (en) * 2017-02-24 2021-10-28 Merck Sharp & Dohme Corp. Enhancing immunogenicity of streptococcus pneumoniae polysaccharide-protein conjugates

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BARALDOI ET AL., INFECT IMMUN, vol. 72, 2004, pages 4884 - 4887
DOUGLAS ET AL., J. BACTERIOL., vol. 169, no. 11, 1987, pages 4967 - 4971
FALUGI ET AL., EUR J IMMUNOL, vol. 31, 2001, pages 3816 - 3824
GIANNINI, G ET AL., NUC. ACIDS RES., vol. 12, no. 10, 1984, pages 4063 - 4069
GROSSMANN LUTZ ET AL: "Current insights into protein solubility: A review of its importance for alternative proteins", FOOD HYDROCOLLOIDS, ELSEVIER BV, NL, vol. 137, 13 December 2022 (2022-12-13), XP087244811, ISSN: 0268-005X, [retrieved on 20221213], DOI: 10.1016/J.FOODHYD.2022.108416 *
KUO ET AL., INFECT IMMUN, vol. 63, 1995, pages 2706 - 2713
NICHOLLSYOULE: "Genetically Engineered Toxins", 1992, MAECEL DEKKER INC.
UCHIDA ET AL., J. BIOL. CHEM., vol. 218, 1973, pages 3838 - 3844
UCHIDA ET AL., NATURE NEW BIOLOGY, vol. 233, 1971, pages 8 - 11

Similar Documents

Publication Publication Date Title
US11020469B2 (en) Immunogenic compositions comprising conjugated capsular saccharide antigens, kits comprising the same and uses thereof
JP7585203B2 (en) Immunogenic multi-heteroantigenic polysaccharide-protein conjugates and uses thereof
EP3576759B1 (en) Methods for production of capsular polysaccharide protein conjugates from streptococcus pneumoniae serotype 19f
US20200405837A1 (en) Immunogenic compositions for use in pneumococcal vaccines
US20220387613A1 (en) Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
US20250381259A1 (en) Immunogenic Compositions Comprising Conjugated Capsular Saccharide Antigens and Uses Thereof
KR20230135178A (en) Neisseria meningitidis vaccine
EP4346893A2 (en) Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
WO2025219904A1 (en) Improved methods for producing glycoconjugates by reductive amination in aprotic solvent
US20090136547A1 (en) Zwitterionization of capsular saccharides
WO2025133971A1 (en) Improved methods for producing bacterial capsular saccharide glycoconjugates
CA3252845A1 (en) Process for producing fertilizer from a biogas stream
RU2854787C2 (en) Immunogenic compositions containing conjugated capsular saccharide antigens, and their use
JP2022075575A (en) Immunogenic compositions for use in pneumococcal vaccines
HK40112911A (en) Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
EP4661911A1 (en) Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
TW202600584A (en) Immunogenic compositions comprising conjugated capsular saccharide antigens and uses thereof
TW202527906A (en) Adjuvanted immunogenic compositions comprising conjugated pneumococcal capsular saccharide antigens and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25724012

Country of ref document: EP

Kind code of ref document: A1