Insulin polypeptide-oligomer conjugates, proinsulin polypeptide-oligomer conjugates and methods of synthesizing same
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
A61K-038/28
C12P-021/06
C07K-005/00
C07K-007/00
C07K-016/00
C07K-017/00
출원번호
US-0389499
(2003-03-14)
등록번호
US-7312192
(2007-12-25)
발명자
/ 주소
Radhakrishnan,Balasingam
Soltero,Richard
Ekwuribe,Nnochiri N.
Puskas,Monica
Sangal,Diti
출원인 / 주소
Biocon Limited
대리인 / 주소
Moore & Van Allen PLLC
인용정보
피인용 횟수 :
0인용 특허 :
135
초록▼
Methods for synthesizing proinsulin polypeptides are described that include contacting a proinsulin polypeptide including an insulin polypeptide coupled to one or more peptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer under conditions sufficient t
Methods for synthesizing proinsulin polypeptides are described that include contacting a proinsulin polypeptide including an insulin polypeptide coupled to one or more peptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide with an oligomer under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate, and cleaving the one or more peptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. Methods of synthesizing proinsulin polypeptide-oligomer conjugates are also provided as are proinsulin polypeptide-oligomer conjugates. Methods of synthesizing C-peptide polypeptide-oligomer conjugates and other pro-polypeptide-oligomer conjugates are also provided.
대표청구항▼
What is claimed is: 1. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide with an oligomer comprising a hydrophilic moiety and/or a lipophilic moiety under conditions sufficient to couple the oligomer to the proinsulin polypeptide
What is claimed is: 1. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide with an oligomer comprising a hydrophilic moiety and/or a lipophilic moiety under conditions sufficient to couple the oligomer to the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate, wherein the proinsulin polypeptide comprises: (i) an insulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide; and (ii) one or more non-insulin polypeptides coupled to the insulin polypeptide by peptide bond(s) capable of being cleaved to yield the insulin polypeptide; and (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. 2. The method of claim 1 wherein: (a) the proinsulin polypeptide comprises multiple conjugation sites; and (b) step 1(a) yields a proinsulin polypeptide-oligomer comprising multiple oligomers. 3. The method of claim 1 wherein: (a) the proinsulin polypeptide comprises one or more conjugation sites on the insulin polypeptide portion thereof, and (b) step 1(a) yields a proinsulin polypeptide-oligomer comprising one or more oligomers on the insulin polypeptide portion thereof. 4. The method of claim 3 wherein step 1 (a) yields a proinsulin polypeptide-oligomer conjugate wherein the non-insulin polypeptide(s) are unconjugated. 5. The method of claim 1 wherein: (a) the proinsulin polypeptide comprises: (i) at least one conjugation site on the insulin polypeptide portion thereof; and (ii) at least one conjugation site on one or more non-insulin polypeptide portions thereof; and (b) step 1 (a) yields a proinsulin polypeptide-oligomer comprising: (i) at least one oligomer coupled to the insulin polypeptide portion thereof; and (ii) at least one oligomer coupled to one or more of the non-insulin polypeptide portion(s) thereof. 6. The method of claim 1, wherein the step 1 (a) comprises: (a) contacting the oligomer with an activating agent under conditions sufficient to provide an activated oligomer capable of coupling to a nucleophilic functionality on the proinsulin polypeptide; and (b) contacting the activated oligomer with the proinsulin polypeptide under conditions sufficient to provide the proinsulin polypeptide-oligomer conjugate. 7. The method of claim 6, wherein the activated group is selected from the group consisting of hydroxysuccinimide, a nucleophilic moiety that couples with lysine, 4-nitrophenyl carbonate, and an activated ester. 8. The method of claim 6, wherein the activated group renders the oligomer capable of covalently coupling to an amino acid functional side chain. 9. The method of claim 6, wherein the activated group renders the oligomer capable of covalently coupling to a hydroxyl functionality on an amino acid or on a modified hydroxyl-modified lysine. 10. The method of claim 6, wherein the activated group renders the oligomer capable of covalently coupling to a carboxylic acid functionality. 11. The method of claim 6, wherein step 6(a) is performed in situ. 12. The method of claim 6, wherein the molar ratio of activated oligomer to proinsulin polypeptide in step 6(b) is greater than about 1:1. 13. The method of claim 6, wherein the molar ratio of activated oligomer to proinsulin polypeptide in step 6(b) is greater than about 3:1. 14. The method of claim 6, wherein the molar ratio of activated oligomer to proinsulin polypeptide in step 6(b) is greater than about 4:1. 15. The method of claim 1, wherein the oligomer comprises a polyethylene glycol moiety. 16. The method of claim 1, wherein the oligomer consists essentially of a polyethylene glycol moiety. 17. The method of claim 1, wherein the oligomer comprises a protected polysaccharide moiety. 18. The method of claim 1, wherein: (a) the insulin polypeptide comprises an A-chain polypeptide and a B-chain polypeptide, and (b) the one or more non-insulin polypeptides comprise a connecting peptide coupled at a first end to the C-terminus of the B-chain polypeptide and coupled at a second end to the N-terminus of the A-chain polypeptide. 19. The method of claim 18 wherein: (a) the B-chain comprises a conjugation site at B29, and (b) the insulin polypeptide-oligomer conjugate is conjugated at the B29 conjugation site. 20. The method of claim 18 wherein: (a) the proinsulin polypeptide has a single lysine at B29, and (b) the insulin polypeptide-oligomer conjugate is a B29 monoconjugate. 21. The method of claim 18 wherein: (a) the proinsulin polypeptide comprises a lysine at B29 and a conjugation site at B1, and (b) the insulin polypeptide-oligomer conjugate is a B1, B29 diconjugate. 22. The method of claim 18, wherein the connecting peptide is a C-peptide polypeptide. 23. The method of claim 22 wherein: (a) the C-peptide comprises a lysine, and (b) step 1(a) yields a proinsulin polypeptide-oligomer in which the lysine(s) of the C-peptide are coupled to oligomer(s). 24. The method of claim 18, wherein the connecting peptide is C-peptide. 25. The method of claim 18, wherein the connecting peptide is devoid of lysine residues. 26. The method of claim 18, wherein the proinsulin polypeptide further comprises a leader peptide coupled to the N-terminus of the B-chain polypeptide. 27. The method of claim 26 wherein: (a) the leader peptide comprises a lysine, and (b) step 1(a) yields a proinsulin polypeptide-oligomer in which the lysine(s) of the leader peptide are coupled to oligomer(s). 28. The method of claim 18, wherein the leader peptide is devoid of lysine residues. 29. The method of claim 26 wherein step 1 (a) yields a proinsulin polypeptide-oligomer comprising an oligomer coupled at an N-terminus of the leader peptide. 30. The method of claim 18, wherein the one or more non-insulin polypeptides further comprise a leader peptide coupled to the N-terminus of the B-chain polypeptide. 31. The method of claim 30 wherein: (a) the leader peptide comprises a lysine, and (b) step 1 (a) yields a proinsulin polypeptide-oligomer in which the lysine(s) of the C-peptide are coupled to oligomer(s). 32. The method of claim 30, wherein the leader peptide is devoid of lysine residues. 33. The method of claim 1, wherein: (a) the insulin polypeptide comprises an A-chain polypeptide and a B-chain polypeptide, and (b) the C-terminus of the B-chain polypeptide is coupled at the N-terminus of the A-chain polypeptide. 34. The method of claim 1, wherein the proinsulin polypeptide is proinsulin. 35. The method of claim 1, wherein the proinsulin polypeptide is proinsulin coupled at the N-terminus of the B-chain to a leader peptide by a cleavable peptide bond. 36. The method of claim 1, wherein the insulin polypeptide is human insulin. 37. The method of claim 36, wherein the oligomer is coupled to the lysine at the B29 position of the insulin. 38. The method of claim 1, wherein the insulin polypeptide is an insulin analog selected from the group consisting of GlyA21 insulin, human;AlaA21 insulin, human;-GlnB3 insulin, human; GlnB30 insulin, human; GlnB3 GluB30 insulin, human; AspB28 insulin, human; LysB28 insulin, human; LeuB28 insulin, human; ValB28 insulin, human; AlaB28 insulin, human; AspB28 ProB29 insulin, human; LysB28 ProB29 insulin, human; LeuB28 ProB29 insulin, human; ValB28 ProB29 insulin, human; and AlaB28 ProB29 insulin, human. 39. The method of claim 1, wherein the insulin polypeptide-oligomer conjugate is amphiphilically balanced. 40. The method of claim 1, wherein the oligomer is present as a substantially monodispersed mixture. 41. The method of claim 1, wherein the oligomer is present as a monodispersed mixture. 42. The method of claim 1, wherein the hydrophilic moiety is a polyalkylene glycol moiety. 43. The method of claim 42, wherein the polyalkylene glycol moiety is a polyethylene glycol moiety. 44. The method of claim 42, wherein the polyalkylene glycol moiety has between 1 and 50 polyalkylene glycol subunits. 45. The method of claim 42, wherein the polyalkylene glycol moiety has between 3 and 50 polyalkylene glycol subunits. 46. The method of claim 42, wherein the polyalkylene glycol moiety has between 2 and 10 polyalkylene glycol subunits. 47. The method of claim 42, wherein the polyalkylene glycol moiety has between 4 and 10 polyalkylene glycol subunits. 48. The method of claim 42, wherein the polyalkylene glycol moiety has at least 2 polyalkylene glycol subunits. 49. The method of claim 1, wherein the lipophilic moiety is an alkyl or fatty acid moiety. 50. The method of claim 1, wherein the lipophilic moiety is a cholesterol or alkyl protected sugar molecule. 51. The method of claim 1, wherein the lipophilic moiety has between 1 and 28 carbon atoms. 52. The method of claim 1, wherein the lipophilic moiety has between 2 and 24 carbon atoms. 53. The method of claim 1, wherein the lipophilic moiety has between 3 and 18 carbon atoms. 54. The method of claim 1, wherein the lipophilic moiety has between 4 and 12 carbon atoms. 55. The method of claim 1, wherein the lipophilic moiety has between 5 and 7 carbon atoms. 56. The method of claim 1, wherein the lipophilic moiety has between 4 and 14 carbon atoms. 57. A method of synthesizing an insulin polypeptide from a proinsulin polypeptide, the method comprising: (a) synthesizing an insulin polypeptide-oligomer conjugate according to claim 1; and (b) hydrolyzing the oligomer(s) from the insulin polypeptide-oligomer conjugate to yield the insulin polypeptide. 58. The method of claim 57 wherein the insulin polypeptide is human insulin. 59. The method of claim 1 wherein the cleaving step cleaves the proinsulin polypeptide-oligomer conjugate at an arginine or lysine. 60. The method of claim 1 wherein the cleaving step cleaves the proinsulin polypeptide-oligomer conjugate at one or more sites comprising a trypsm cleavage site. 61. The method of claim 1 wherein the cleaving step cleaves the proinsulin polypeptide-oligomer conjugate at one or more sites comprising an arginine cleavage site. 62. The method of claim 1, wherein the cleaving of the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate comprises contacting the proinsulin polypeptide-oligomer conjugate with one or more enzymes that are capable of cleaving the bond(s) between the one or more non-insulin polypeptides and the insulin polypeptide under conditions sufficient to cleave the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate. 63. The method of claim 62, wherein the one or more enzymes are selected from the group consisting of trypsin, carboxy peptidase B, and mixtures thereof. 64. The method of claim 18, wherein the connecting peptide has a terminal amino acid residue at the first end, and wherein the cleaving of the connecting peptide from the proinsulin polypeptide-oligomer conjugate comprises: (a) contacting the proinsulin polypeptide-oligomer conjugate with a first enzyme under conditions sufficient to provide a terminal amino acid residue-insulin polypeptide-oligomer conjugate; and (b) contacting the terminal amino acid residue-insulin polypeptide-oligomer conjugate with a second enzyme under conditions sufficient to provide the insulin polypeptide-oligomer conjugate. 65. The method of claim 64, wherein the terminal amino acid residue is an arginine, proline or lysine residue. 66. The method of claim 65, wherein the insulin polypeptide is human insulin, and wherein the connecting peptide is human C-peptide. 67. The method of claim 64, wherein the contacting of the proinsulin polypeptide-oligomer conjugate with a first enzyme and the contacting of the terminal amino acid residue-insulin polypeptide-oligomer conjugate with a second enzyme occur substantially concurrently. 68. The method of claim 67, wherein the first enzyme and the second enzyme are provided in a mixture comprising the first enzyme and the second enzyme. 69. The method of claim 64, wherein the first enzyme is trypsin, and wherein the second enzyme is carboxy peptidase B. 70. The method of claim 1, further comprising chemically modifying one or more of the oligomer(s) of the insulin polypeptide-oligomer conjugate. 71. The method of claim 1, further comprising activating one or more of the oligomer(s) of the insulin polypeptide-oligomer conjugate. 72. The method of claim 1, further comprising lengthening one or more of the oligomer(s) of the insulin polypeptide-oligomer conjugate. 73. The method of claim 1, further comprising shortening one or more of the oligomer(s) of the insulin polypeptide-oligomer conjugate. 74. The method of claim 1, wherein the yield of insulin polypeptide-oligomer conjugate is greater than 75 percent. 75. The method of claim 1, wherein the yield of insulin polypeptide-oligomer conjugate is greater than 85 percent. 76. The method of claim 1, wherein the yield of insulin polypeptide-oligomer conjugate is greater than about 90 percent. 77. The method of claim 1, wherein the yield of insulin polypeptide-oligomer conjugate is greater than 95 percent. 78. The method of claim 1, wherein the yield of insulin polypeptide-oligomer conjugate is greater than 99 percent. 79. A method of synthesizing an insulin molecule, comprising synthesizing an insulin polypeptide-oligomer conjugate according to claim 1, and hydrolyzing the oligomer(s) from the polypeptide-oligomer conjugate to yield the insulin molecule. 80. A method of synthesizing insulin, comprising synthesizing an insulin polypeptide-oligomer conjugate according to claim 1, and hydrolyzing the oligomer(s) from the polypeptide-oligomer conjugate to yield insulin. 81. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide comprising an insulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide, the insulin polypeptide coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide, with an oligomer comprising the structure of Formula I: description="In-line Formulae" end="lead"A-Lj-Gk-R-G' m-R'-G"n-T (I)description="In-line Formulae" end="tail" wherein: A is an activatable moiety; L is an optional linker moiety; G, G' and G" are each optional spacer moieties; R is a lipophilic moiety and R' is a polyalkylene glycol moiety, or R' is the lipophilic moiety and R is the polyalkylene glycol moiety, and wherein, (i) R and R' are both present, or (ii) R and G are absent and L is coupled to G' if present or to R' if G' is not present, or (iii) R' and G" are absent and T is coupled to G' if present or to R if G' is not present; T is a terminating moiety; and j, k, m and n are individually 0 or 1; under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate; and (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. 82. The method of claim 81, wherein R and R' are both present. 83. The method of claim 81, wherein R and G are absent and L is coupled to G' if present or to R' if G' is not present. 84. The method of claim 81, wherein R' is a polyethylene glycol moiety. 85. The method of claim 81, wherein R' and G" are absent and T is coupled to G' if present or to R if G' is not present. 86. The method of claim 81, wherein A is selected from the group consisting of--C(O)--OH, C(S)--OH,--C(S)--SH,--OH,--SH, and NH2. 87. The method of claim 81, wherein L is selected from the group consisting of alkyl moieties and fatty acid moieties. 88. The method of claim 81, wherein G, G' and G" are individually selected from the group consisting of sugar moieties, cholesterol, and glycerine moieties. 89. The method of claim 81, wherein T is selected from the group consisting of alkyl and alkoxy. 90. The method of claim 81, wherein: A is a carboxylic acid moiety; R is an alkyl moiety having between 3 and 8 carbon atoms; R' is polyethylene glycol having between 4 and 10 polyethylene glycol subunits; T is lower alkyl or lower alkoxy; and j, k, m and n are 0. 91. The method of claim 81, wherein: A is a carboxylic acid moiety; R is an alkyl moiety having between 3 and 8 carbon atoms; R' is polyethylene glycol having 7 polyethylene glycol subunits; T is methoxy; and j, k, m and n are 0. 92. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide comprising an insulin polypeptide, the insulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide and coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide, with an activated form of an oligomer comprising the structure of Formula II: description="In-line Formulae" end="lead"A-X(CH2)mY(C2H4O)n R (II)description="In-line Formulae" end="tail" wherein: A is--C(O)--OH, C(S)--OH,--C(S)--SH,--OH,--SH, or NH2; X is an oxygen atom or a covalent bond, with the proviso that X is not an oxygen atom when A is--OH; Y is an ester, an ether, a carbamate, a carbonate, or an amide bonding moiety; m is between 0 and 30; n is between 0 and 50; m and n are not both 0; and R is an alkyl moiety, a sugar moiety, cholesterol, adamantane, an alcohol moiety, or a fatty acid moiety; under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate; and (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. 93. The method of claim 92 wherein m is between 3 and 16. 94. The method of claim 92, wherein m is between 4 and 14. 95. The method of claim 92, wherein m is between 5 and 10. 96. The method of claim 92, wherein n is between 3 and 18. 97. The method of claim 92, wherein n is between 4 and 14. 98. The method of claim 92, wherein n is between 5 and 10. 99. The method of claim 92, wherein R is lower alkyl. 100. The method of claim 92, wherein R is C1 to C3 alkyl. 101. The method of claim 92, wherein R is methyl. 102. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide comprising an insulin polypeptide, the insulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide and coupled to one or more non-insulin polypeptides by peptide bond(s) capable of being cleaved to yield the insulin polypeptide, with an activated form of an oligomer comprising the structure of Formula III: description="In-line Formulae" end="lead"A-(CH2)m(OC2H4)n OR (III)description="In-line Formulae" end="tail" wherein: A is--C(O)--OH, C(S)--OH,--C(S)--SH,--OH,--SH, or NH2; m is between 0 and 25; n is between 0 and 25; m and n are not both 0; and R is alkyl; under conditions sufficient to couple the oligomer to the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate; and (b) cleaving the one or more non-insulin polypeptides from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. 103. The method of claim 102, wherein m is between 3 and 16. 104. The method of claim 102, wherein m is between 4 and 14. 105. The method of claim 102, wherein m is between 5 and 10. 106. The method of claim 102, wherein n is between 3 and 18. 107. The method of claim 102, wherein n is between 4 and 14. 108. The method of claim 102, wherein n is between 5 and 10. 109. The method of claim 102, wherein R is lower alkyl. 110. The method of claim 102, wherein R is C1 to C3 alkyl. 111. The method of claim 102, wherein R is methyl. 112. A method of synthesizing an insulin polypeptide-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide comprising: (i) an insulin polypeptide, the insulin polypeptide having a human A-chain polypeptide and a human B-chain polypeptide, which human B-chain polypeptide comprises a lysine residue; (ii) a connecting peptide coupled at a first end to the C-terminus of the human B-chain polypeptide and coupled at a second end to the N-terminus of the human A-chain polypeptide; and (iii) a leader peptide coupled to the N-terminus of the human B-chain polypeptide with an activated form of an oligomer comprising the structure of Formula IV: wherein: m is between 0 and 30; n is between 0 and 50; m and n are not both 0; and R is alkyl; under conditions sufficient to couple the oligomer to the lysine residue of the human B-chain polypeptide of the insulin polypeptide portion of the proinsulin polypeptide and provide a proinsulin polypeptide-oligomer conjugate; and (b) enzymatically cleaving the connecting peptide and the leader peptide from the proinsulin polypeptide-oligomer conjugate to provide the insulin polypeptide-oligomer conjugate. 113. The method of claim 112, wherein m is between 3 and 16. 114. The method of claim 112, wherein m is between 4 and 14. 115. The method of claim 112, wherein m is between 5 and 10. 116. The method of claim 112, wherein n is between 3 and 18. 117. The method of claim 112, wherein n is between 4 and 14. 118. The method of claim 112, wherein n is between 5 and 10. 119. The method of claim 112, wherein R is lower alkyl. 120. The method of claim 112, wherein R is C1 to C3 alkyl. 121. The method of claim 112, wherein R is methyl. 122. A method of synthesizing an insulin-oligomer conjugate comprising: (a) contacting a proinsulin polypeptide, which comprises an insulin polypeptide, the insulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide and coupled at its N-terminus to a leader peptide, with an activated form of an oligomer comprising the structure of Formula V: under conditions sufficient to couple the oligomer to the B29 lysine residue of the proinsulin and provide a proinsulin polypeptide-oligomer conjugate; and (b) enzymatically cleaving a C-peptide and the leader peptide from the proinsulin polypeptide-oligomer conjugate to provide the insulin-oligomer conjugate. 123. The method of claim 122, wherein the enzymatically cleaving of the C-peptide and the leader peptide from the proinsulin polypeptide-oligomer conjugate comprises: (a) contacting the proinsulin polypeptide-oligomer conjugate with a first enzyme under conditions sufficient to provide an (Arg31)-insulin-oligomer conjugate; and (b) contacting the (Arg31)-insulin polypeptide-oligomer conjugate with a second enzyme under conditions sufficient to provide the insulin polypeptide-oligomer conjugate. 124. The method of claim 123, wherein the first enzyme is trypsin. 125. The method of claim 123, wherein the second enzyme is carboxy peptidase B. 126. A method of synthesizing a human insulin polypeptide-acyl oligomer conjugate comprising enzymatically cleaving one or more non-insulin polypeptides from a proinsulin polypeptide-acyl oligomer conjugate to provide the human insulin polypeptide-acyl oligomer conjugate. 127. A method of synthesizing an insulin-acyl oligomer conjugate comprising enzymatically cleaving a leader peptide and a C-peptide from a proinsulin polypeptide-acyl oligomer conjugate comprising the following structure: to provide the insulin-acyl oligomer conjugate comprising the following structure: wherein Insulin is human insulin. 128. The method of claim 127, wherein the leader peptide is devoid of lysine residues. 129. The method of claim 127, wherein the enzymatically cleaving of the C-peptide and the leader peptide from the proinsulin polypeptide-acyl oligomer conjugate comprises: (a) contacting the proinsulin polypeptide-oligomer conjugate with a first enzyme under conditions sufficient to provide an (Arg31)-insulin-oligomer conjugate; and (b) contacting the (Arg31)-insulin polypeptide-oligomer conjugate with a second enzyme under conditions sufficient to provide the insulin polypeptide-oligomer conjugate. 130. The method of claim 127, wherein the first enzyme comprises a trypsin-like enzyme. 131. The method of claim 127, wherein the first enzyme comprises trypsin and/or a peptidase of Achromobacter lyticus and/or thrombin. 132. The method of claim 127, wherein the second enzyme comprises a carboxypeptidase-like enzyme. 133. The method of claim 127, wherein the second enzyme comprises a carboxypeptidase A and/or B. 134. A method of synthesizing a proinsulin polypeptide-oligomer conjugate, the proinsulin polypeptide comprising a human A-chain polypeptide and a human B-chain polypeptide, the method comprising contacting a proinsulin polypeptide with an oligomer comprising a hydrophilic moiety and a lipophilic moiety under conditions sufficient to provide the proinsulin polypeptide-oligomer conjugate. 135. A method of synthesizing a C-peptide polypeptide-oligomer conjugate comprising: (a) contacting a pro-C-peptide polypeptide comprising a human C-peptide polypeptide coupled to one or more non-insulin polypeptides by peptide bond(s) that are cleavable to yield the human C-peptide polypeptide with an oligomer under conditions sufficient to couple the oligomer to the human C-peptide polypeptide portion of the pro-C-peptide polypeptide and provide a pro-C-peptide polypeptide-oligomer conjugate; and (b) cleaving the one or more non-insulin polypeptides from the pro-C-peptide polypeptide-oligomer conjugate to provide the human C-peptide polypeptide-oligomer conjugate. 136. The method of claim 135, wherein the pro-C-peptide polypeptide is a proinsulin polypeptide. 137. The method of claim 135, wherein the pro-C-peptide polypeptide is proinsulin.
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Brownlee Michael (Watertown MA) Cerami Anthony (Flanders NJ), Method and system for the controlled release of biologically active substances to a body fluid.
Brownlee Michael (Watertown MA) Cerami Anthony (Flanders NJ), Method and system for the controlled release of biologically active substances to a body fluid.
Rhee Woonza (Palo Alto CA) Wallace Donald G. (Menlo Park CA) Michaels Alan S. (Boston MA) Burns ; Jr. Ramon A. (Fremont CA) Fries Louis (Los Altos CA) DeLustro Frank (Belmont CA) Bentz Hanne (Newark , Method of preparing collagen-polymer conjugates.
Harris J. Milton ; Veronese Francesco Maria,ITX ; Caliceti Paolo,ITX ; Schiavon Oddone,ITX, Multiarmed, monofunctional, polymer for coupling to molecules and surfaces.
Davis Frank F. (19 Farmingdale Rd. East Brunswick NJ 08816) Van Es Theodorus (313 Overbrook Rd. Piscataway NJ 08854) Palczuk Nicholas C. (45 W. Franklin St. Bound Brook NJ 08805), Non-immunogenic polypeptides.
Rhee Woonza (Palo Alto CA) Wallace Donald G. (Menlo Park CA) Michaels Alan S. (Boston MA) Burns ; Jr. Ramon A. (Fremont CA) Fries Louis (Los Altos CA) DeLustro Frank (Belmont CA) Bentz Hanne (Newark , Polymer conjugates ophthalmic devices comprising collagen-polymer conjugates.
Nitecki Danute E. (Berkeley CA) Katre Nandini (El Cerrito CA) Goodson Robert J. (Albany CA) Aldwin Lois (San Mateo CA), Preparation of a polymer/interleukin-2 conjugate.
Baker Jeffrey Clayton (Indianapolis IN) Moser Brian A. (Indianapolis IN) Shrader Warren E. (Indianapolis IN), Preparation of an acylated protein powder.
Baker Jeffrey C. (Indianapolis IN) Carter Nancy D. (Indianapolis IN) Frank Bruce H. (Indianapolis IN), Preparation of stable zinc insulin analog crystals.
Appelgren Curt H. (Kungsbacka SEX) Eskilsson Eva C. (Mlnlycke SEX) Uvdal Jonas P. (Gothenburg SEX), Process for preparing rapidly disintegrating granulates.
Maitra Amarnath,INX ; Ghosh Prashant Kumar,INX ; De Tapas K.,INX ; Sahoo Sanjeeb Kumar,INX, Process for the preparation of highly monodispersed polymeric hydrophilic nanoparticles.
Frank Bruce H. (Indianapolis IN) Prouty Walker E. (Indianapolis IN) Heiney Richard E. (Greenwood IN) Walden Mark R. (Indianapolis IN), Process for transforming a human insulin precursor to human insulin.
Backstrom Kjell Goran Erik,SEX ; Dahlback Carl Magnus Olof,SEX ; Edman Peter,SEX ; Johansson Ann Charlotte Birgit,SEX, Processes for preparing compositions for inhalation.
Eckenhoff James B. (Los Altos CA) Cortese Richard (Los Gatos CA) Landrau Felix A. (Milpitas CA), Rate controlled dispenser for administering beneficial agent.
Baker Jeffrey C. (Indianapolis IN) Hanquier Jose M. (Martinsville IN) Shrader Warren E. (Indianapolis IN), Reducing gelation of a fatty acid-acylated protein.
Baker Jeffrey C. (Indianapolis IN) Chen Victor J. (Indianapolis IN) Hanquier Jose M. (Martinsville IN) Kriauciunas Aidas (Indianapolis IN) Moser Brian A. (Indianapolis IN) Shuman Robert T. (Greenwood, Selective acylation of epsilon-amino groups.
Backstrom Kjell Goran Erik,SEX ; Dahlback Carl Magnus Olof,SEX ; Edman Peter,SEX ; Johansson Ann Charlotte Birgit,SEX, Systemic administration of a therapeutic preparation.
Bckstrm Kjell G. E. (Lund SEX) Dahlbck Carl M. O. (Lund SEX) Edman Peter (Bjrred SEX) Johansson Ann C. B. (Lund SEX), Systemic administration of a therapeutic preparation.
Backstrom Kjell Goran Erik (Lund SEX) Dahlback Carl Magnus Olof (Lund SEX) Edman Peter (Bjarred SEX) Johansson Ann Charlotte Birgit (Lund SEX), Therapeutic preparation for inhalation.
Backstrom Kjell Goran Erik,SEX ; Dahlback Carl Magnus Olof,SEX ; Edman Peter,SEX ; Johansson Ann Charlotte Birgit,SEX, Therapeutic preparation for inhalation.
Backstrom Kjell Goran Erik,SEX ; Dahlback Carl Magnus Olof,SEX ; Edman Peter,SEX ; Johansson Ann Charlotte Birgit,SEX, Therapeutic preparation for inhalation.
Backstrom Kjell Goran Erik,SEX ; Dahlback Carl Magnus Olof,SEX ; Edman Peter,SEX ; Johansson Ann Charlotte Birgit,SEX, Therapeutic preparation for inhalation.
Bckstrm Kjell G. E. (Lund SEX) Dahlbck Carl M. O. (Lund SEX) Edman Peter (Bjrred SEX) Johansson Ann C. B. (Lund SEX), Therapeutic preparation for inhalation.
Longenecker John P. (Mountain View CA) Ennis Richard (Fremont CA) Baldwin Patricia A. (Hayward CA) Lee William A. (Los Altos CA), Transmembrane formulations for drug administration.
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