Soy Peptone as a Nitrogen Source in Preparing Meningcoccal Conjugates
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61K-039/095
C12P-019/04
C12P-019/00
C12N-001/20
출원번호
US-0587174
(2005-04-22)
공개번호
US-0160044
(2008-07-03)
우선권정보
GB-0408978.5(2004-04-22)
국제출원번호
PCT/GB05/001543
(2005-04-22)
발명자
/ 주소
Marshall,Cameron John
출원인 / 주소
CHIRON SRL
대리인 / 주소
NOVARTIS VACCINES AND DIAGNOSTICS INC.
인용정보
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0인용 특허 :
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초록▼
A method for preparing a protein-saccharide conjugate, comprising the steps of: (a) preparing an aqueous growth medium comprising soy peptone as a nitrogen source; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d) prep
A method for preparing a protein-saccharide conjugate, comprising the steps of: (a) preparing an aqueous growth medium comprising soy peptone as a nitrogen source; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d) preparing capsular saccharide from the bacterium; and (e) conjugating the capsular saccharide to a carrier protein, to give the protein-saccharide conjugate is disclosed. The conjugates are useful in vaccine production.
대표청구항▼
1. A method for preparing a protein-saccharide conjugate, comprising the steps of: (a) preparing an aqueous growth medium comprising soy peptone as a nitrogen source; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d)
1. A method for preparing a protein-saccharide conjugate, comprising the steps of: (a) preparing an aqueous growth medium comprising soy peptone as a nitrogen source; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d) preparing capsular saccharide from the bacterium; and (e) conjugating the capsular saccharide to a carrier protein, to give the a protein-saccharide conjugate. 2. The method of claim 1, comprising the steps of: (a) preparing an aqueous growth medium comprising: (i) between 1 and 5 g/l sodium phosphate, dibasic; (ii) between 1 and 50 g/l soy peptone; (iii) between 2 and 10 g/l monosodium glutamate; (iv) between 20 and 200 mg/l potassium chloride; (v) between 500 and 1000 mg/l magnesium sulfate; (vi) between 5 and 20 g/l glucose; and (vii) between 5 and 30 mg/l L-cysteine; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d) preparing capsular saccharide from the bacterium; and (e) conjugating the capsular saccharide to a carrier protein, to give the a protein-saccharide conjugate. 3. The method of claim 2, wherein the medium comprises: (i) 2.5 g/l sodium phosphate, dibasic; (ii) between 5 and 30 g/l soy peptone; (iii) 5 g/l monosodium glutamate; (iv) 0.103 g/l potassium chloride; (v) 0.732 g/l magnesium sulfate; (vi) 11.250 g/l glucose; and, optionally, (vii) 0.016 g/l L-cysteine. 4. The method of claim 1, comprising the steps of: (a) preparing an aqueous growth medium comprising: (i) between 2 and 20 g/l glucose; (ii) between 1 and 50 g/l soy peptone; (iii) between 2 and 10 g/l sodium chloride; (iv) between 0.2 and 4 g/l potassium sulfate; (v) between 1 and 10 g/l potassium phosphate, dibasic; (vi) between 50 and 400 mg/l magnesium chloride; (vii) between 5 and 50 mg/l calcium chloride; (viii) between 0.5 and 5 mg/l ferrous sulfate; and, optionally. L-amino acids, such that the medium comprises: between 1 and 10 g/l L-glutamic acid, between 0.1 and 5 g/l L-arginine, between 0.1 and 5 g/l L-serine and/or between 0.05 and 0.5 g/l L-cysteine; (b) inoculating the medium with a Neisseria meningitidis bacterium; (c) incubating the medium to allow growth of the bacterium; (d) preparing capsular saccharide from the bacterium; and (e) conjugating the capsular saccharide to a carrier protein, to give the a protein-saccharide conjugate. 5. The method of claim 4, wherein the medium comprises: (i) 10 g/l glucose; (ii) between 5 and 30 g/l soy peptone; (iii) 5.80 g/l sodium chloride; (iv) 1 g/l potassium sulfate; (v) 4 g/l potassium phosphate, dibasic; (vi) 0.19 g/l magnesium chloride; (vii) 0.021 g/l calcium chloride; (viii) 0.002 g/l ferrous sulfate; and, optionally, a mixture of amino acids comprising 5 g/l L-glutamic acid, 0.3 g/l L-arginine, 0.5 g/l L-serine and 0.23 g/l L-cysteine. 6. The method of claim 1, wherein the medium includes a foam control agent. 7. The method of claim 1, wherein the medium does not include ammonium chloride. 8. The method of claim 1, wherein steps (b) and (c) are repeated more than once, with inoculation into fresh medium in each repeat. 9. The method of claim 1, wherein step (c) takes place at 30-40�� C. 10. The method of claim 1, wherein step (c) comprises fed-batch culture. 11. The method of claim 10, wherein the fed-batch culture is def with a feed solution that comprises: 50 g/l glucose; 50 g/l glutamic acid; 3 g/l arginine; 3 g/l serine; 2 g/l cysteine; 10 g/l NH4Cl; 2 g/l MgCl2; 0.14 g/l CaCl2; and 0.02 g/l FeSO4. 12. The method of claim 1, wherein step (d) comprises: CTAB addition, centrifugation, and collection of supernatant. 13. The method of claim 1, wherein step (e) comprises conjugation of the saccharide to a diphtheria toxoid carrier protein. 14. The method of claim 1, wherein step (e) comprises: reacting the saccharide with adipic acid dihydrazide; addition of sodium cyanoborohydride; and addition of the carrier protein. 15. The method of claim 1, wherein, between steps (d) and (e), the saccharide is treated with hydrogen peroxide to reduce its chain length. 16. The method of claim 1, wherein the Neisseria meningitidis is serogroup A. 17. The method of claim 1, wherein the Neisseria meningitidis is serogroup C. 18. The method of claim 1, wherein the Neisseria meningitidis is serogroup W135. 19. The method of claim 1, wherein the Neisseria meningitidis is serogroup Y. 20. A method for preparing a mixture of conjugates of the capsular saccharides of serogroups A, C, W135 and Y, comprising the steps of: preparing a conjugate by the method of claim 16; preparing a conjugate by the method of claim 17; preparing a conjugate by the method of claim 18; preparing a conjugate by the method of claim 19; and mixing these four conjugates. 21. The method of claim 20, wherein the four conjugates are mixed with a phosphate buffered saline solution. 22. The method of claim 20, wherein the four conjugates are mixed with an aluminium hydroxide adjuvant. 23. The method of claim 20, wherein the four conjugates are mixed with an aluminium phosphate adjuvant. 24. A method for preparing a pharmaceutical composition, comprising the steps of: (a) preparing a conjugate by the method of claim 1; and (b) mixing the conjugate(s) with one or more pharmaceutically acceptable carriers. 25. The method of claim 24, wherein the pharmaceutical composition is packaged for injection. 26. The method of claim 24, further comprising the step of (c) putting the pharmaceutical composition into a syringe. 27. The method of claim 1, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 28. A feed solution for use during meningococcal culture comprises: 50 g/l glucose; 50 g/l L-glutamic acid; 3 g/l L-arginine; 3 g/l L-serine; 2 g/l L-cysteine; 10 g/l NH4Cl; 2 g/l MgCl2; 0.14 g/l CaCl2; and 0.02 g/l FeSO4. 29. The medium as defined in 1 to 5 claim 1, for use in growing serogroup B of N. meningitidis, or in growing N. gonorrhoeae. 30. The method of claim 2, wherein the medium includes a foam control agent. 31. The method of claim 3, wherein the medium includes a foam control agent. 32. The method of claim 4, wherein the medium includes a foam control agent. 33. The method of claim 5, wherein the medium includes a foam control agent. 34. The method of claim 2, wherein the medium does not include ammonium chloride. 35. The method of claim 3, wherein the medium does not include ammonium chloride. 36. The method of claim 4, wherein the medium does not include ammonium chloride. 37. The method of claim 5, wherein the medium does not include ammonium chloride. 38. The method of claim 2, wherein steps (b) and (c) are repeated more than once, with inoculation into fresh medium in each repeat. 39. The method of claim 3, wherein steps (b) and (c) are repeated more than once, with inoculation into fresh medium in each repeat. 40. The method of claim 4, wherein steps (b) and (c) are repeated more than once, with inoculation into fresh medium in each repeat. 41. The method of claim 5, wherein steps (b) and (c) are repeated more than once, with inoculation into fresh medium in each repeat. 42. The method of claim 2, wherein step (c) takes place at 30-40�� C. 43. The method of claim 3, wherein step (c) takes place at 30-40�� C. 44. The method of claim 4, wherein step (c) takes place at 30-40�� C. 45. The method of claim 5, wherein step (c) takes place at 30-40�� C. 46. The method of claim 2, wherein step (c) comprises fed-batch culture. 47. The method of claim 3, wherein step (c) comprises fed-batch culture. 48. The method of claim 4, wherein step (c) comprises fed-batch culture. 49. The method of claim 5, wherein step (c) comprises fed-batch culture. 50. The method of claim 2, wherein step (d) comprises: CTAB addition, centrifugation, and collection of supernatant. 51. The method of claim 3, wherein step (d) comprises: CTAB addition, centrifugation, and collection of supernatant. 52. The method of claim 4, wherein step (d) comprises: CTAB addition, centrifugation, and collection of supernatant. 53. The method of claim 5, wherein step (d) comprises: CTAB addition, centrifugation, and collection of supernatant. 54. The method of claim 2, wherein step (e) comprises conjugation of the saccharide to a diphtheria toxoid carrier protein. 55. The method of claim 3, wherein step (e) comprises conjugation of the saccharide to a diphtheria toxoid carrier protein. 56. The method of claim 4, wherein step (e) comprises conjugation of the saccharide to a diphtheria toxoid carrier protein. 57. The method of claim 5, wherein step (e) comprises conjugation of the saccharide to a diphtheria toxoid carrier protein. 58. The method of claim 2, wherein step (e) comprises: reacting the saccharide with adipic acid dihydrazide; addition of sodium cyanoborohydride; and addition of the carrier protein. 59. The method of claim 3, wherein step (e) comprises: reacting the saccharide with adipic acid dihydrazide; addition of sodium cyanoborohydride; and addition of the carrier protein. 60. The method of claim 4, wherein step (e) comprises: reacting the saccharide with adipic acid dihydrazide; addition of sodium cyanoborohydride; and addition of the carrier protein. 61. The method of claim 5, wherein step (e) comprises: reacting the saccharide with adipic acid dihydrazide; addition of sodium cyanoborohydride; and addition of the carrier protein. 62. The method of claim 2, wherein, between steps (d) and (e), the saccharide is treated with hydrogen peroxide to reduce its chain length. 63. The method of claim 3, wherein, between steps (d) and (e), the saccharide is treated with hydrogen peroxide to reduce its chain length. 64. The method of claim 4, wherein, between steps (d) and (e), the saccharide is treated with hydrogen peroxide to reduce its chain length. 65. The method of claim 5, wherein, between steps (d) and (e), the saccharide is treated with hydrogen peroxide to reduce its chain length. 66. The method of claim 2, wherein the Neisseria meningitidis is serogroup A. 67. The method of claim 3, wherein the Neisseria meningitidis is serogroup A. 68. The method of claim 4, wherein the Neisseria meningitidis is serogroup A. 69. The method of claim 5, wherein the Neisseria meningitidis is serogroup A. 70. The method of claim 2, wherein the Neisseria meningitidis is serogroup C. 71. The method of claim 3, wherein the Neisseria meningitidis is serogroup C. 72. The method of claim 4, wherein the Neisseria meningitidis is serogroup C. 73. The method of claim 5, wherein the Neisseria meningitidis is serogroup C. 74. The method of claim 2, wherein the Neisseria meningitidis is serogroup W135. 75. The method of claim 3, wherein the Neisseria meningitidis is serogroup W135. 76. The method of claim 4, wherein the Neisseria meningitidis is serogroup W135. 77. The method of claim 5, wherein the Neisseria meningitidis is serogroup W135. 78. The method of claim 2, wherein the Neisseria meningitidis is serogroup Y. 79. The method of claim 3, wherein the Neisseria meningitidis is serogroup Y. 80. The method of claim 4, wherein the Neisseria meningitidis is serogroup Y. 81. The method of claim 5, wherein the Neisseria meningitidis is serogroup Y. 82. The method of claim 21, wherein the four conjugates are mixed with an aluminium hydroxide adjuvant. 83. The method of claim 21, wherein the four conjugates are mixed with an aluminium phosphate adjuvant. 84. A method for preparing a pharmaceutical composition, comprising the steps of: (a) preparing a conjugate by the method of claim 2; and (b) mixing the conjugate(s) with one or more pharmaceutically acceptable carriers. 85. A method for preparing a pharmaceutical composition, comprising the steps of: (a) preparing a conjugate by the method of claim 4; and (b) mixing the conjugate(s) with one or more pharmaceutically acceptable carriers. 86. A method for preparing a pharmaceutical composition, comprising the steps of: (a) preparing a combination of conjugates by the method of claim 20; and (b) mixing the conjugate(s) with one or more pharmaceutically acceptable carriers. 87. The method of claim 84, wherein the pharmaceutical composition is packaged for injection. 88. The method of claim 85, wherein the pharmaceutical composition is packaged for injection. 89. The method of claim 86, wherein the pharmaceutical composition is packaged for injection. 90. The method of claim 25, further comprising the step of (c) putting the pharmaceutical composition into a syringe. 91. The method of claim 87, further comprising the step of (c) putting the pharmaceutical composition into a syringe. 92. The method of claim 88, further comprising the step of (c) putting the pharmaceutical composition into a syringe. 93. The method of claim 89, further comprising the step of (c) putting the pharmaceutical composition into a syringe. 94. The method of claim 2, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 95. The method of claim 4, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 96. The method of claim 20, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 97. The method of claim 24, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 98. The method of claim 84, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 99. The method of claim 85, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 100. The method of claim 86, further comprising the step of mixing a meningococcal conjugate with (a) a Haemophilus influenzae type B capsular saccharide conjugate, and/or (b) a Streptococcus pneumoniae capsular saccharide conjugate. 101. The medium as defined in claim 2, for use in growing serogroup B of N. meningitidis, or in growing N. gonorrhoeae. 102. The medium as defined in claim 3, for use in growing serogroup B of N. meningitidis, or in growing N. gonorrhoeae. 103. The medium as defined in claim 4, for use in growing serogroup B of N. meningitidis, or in growing N. gonorrhoeae. 104. The medium as defined in claim 5, for use in growing serogroup B of N. meningitidis, or in growing N. gonorrhoeae.
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