Method for the crystallization of human serum albumin

• US 20060178309A1
• Filed: 03/17/2006
• Published: 08/10/2006
• Est. Priority Date: 11/19/2002
• Status: Abandoned Application

• Current Assignee: Gtc-Biotherapeutics Incorporated
• Original Assignee: Gtc-Biotherapeutics Incorporated
• Inventors: Couto, Daniel E., Fulton, Scott P., Uotila, Sinikka, Visuri, Kalevi
• Application Number:
  US11/378,727
• Publication Number:
  US 20060178309A1
• Time in Patent Office: Days
• US Class Current: 514/15.200
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/16 Central respiratory analeptics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/08 Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock artificial tears A61P27/04
• CPC Class Codes: A61P 7/10 Antioedematous agents; Diuretics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: Y10S 530/829 Blood
• CPC Class Codes: Y10S 530/83 Plasma; serum

Abstract

The present invention relates to the purification and production of human albumin from various sources through crystallization and repeated crystallization. Basic features of the invented process include providing specific reaction conditions and precipitating reagents to maximize albumin crystallization. Solubility diagrams are utilized as the basis for process control of the invented method. The current invention specifically controls phosphate concentration, pH and temperature to precisely guide crystallization kinetics and crystal yield.

Claims

1. A crystalline human albumin product comprising at least a portion of the material resulting from a process of:

a) concentrating an albumin containing fluid until said fluid has at least 15 grams of albumin per liter of solution;

b) adding a first phosphate mixture to said albumin containing fluid until the concentration of said phosphate mixture is in the range of 2.4-2.6 molar;

c) providing a first filtering of said albumin containing solution so as to form a resultant crystallizing batch solution to remove impurities;

d) cooling the resultant filtrate of said crystallizing batch solution to a temperature of at most 15°

C.;

e) allowing the human albumin in said crystallizing batch solution to crystallize;

f) adding more of said first phosphate mixture to said crystallizing batch solution sufficient to achieve a concentration of at most 3.0 molar;

g) making a first separation of albumin crystals from any remaining fluid;

h) suspending the albumin crystals from said first separation of albumin crystals in a second phosphate mixture wherein said second phosphate mixture has a concentration of 2.7-3.0 M;

i) heating the crystal suspension from said first separation of albumin crystals up to temperature in the range of 40-50°

C. in order to dissolve the crystals,

j) providing a second filtering to the dissolved crystal suspension from said first separation of albumin crystals;

k) cooling the resultant dissolved crystal suspension from said first separation of albumin crystals to a temperature of at most 15°

C.; and

l) allowing albumin crystals to form the resultant cooled crystal suspension;

wherein the application of these specific steps allows the purification and crystallization of human albumin from a given human albumin containing feedstream.

2. The process of claim 1, wherein said first phosphate mixture is comprised of a sodium phosphate salt.

3. The process of claim 1, wherein said first phosphate mixture is a potassium phosphate salt.

4. The process of claim 1, wherein said first phosphate mixture is comprised of both a sodium and a potassium salt.

5. The process of claim 1, wherein said albumin containing fluid has a concentration of albumin in one liter of solution in a range of 15-50 grams.

6. The process of claim 1, wherein the temperature of said first phosphate mixture at the time of addition is in the range of 20-30°

C.

7. The process of claim 1, wherein said first phosphate mixture at the time of addition has a pH in the range of pH 6.0-6.7.

8. The process of claim 1, wherein said filtrate collected after filtering said crystallizing batch is cooled to a temperature of at most 10°

C.

9. The process of claim 1, wherein said crystallizing batch solution is allowed to crystallize for at most 12 hours.

10. The process of claim 1, wherein said crystallizing batch solution is allowed to crystallize for at most 24 hours.

11. The process of claim 1, wherein said crystallizing batch solution is allowed to crystallize for at least 24 hours

12. The process of claim 1, wherein said first separation of albumin crystals is accomplished by filtration.

13. The process of claim 1, wherein said first separation of albumin crystals is accomplished by centrifugation.

14. The process of claim 1, wherein said first separation of albumin crystals is accomplished by gravity.

15. The process of claim 1, wherein said first separation of albumin crystals is accomplished by drying.

16. The process of claim 1, wherein said second phosphate mixture is comprised of a sodium phosphate salt.

17. The process of claim 1, wherein said second phosphate mixture is a potassium phosphate salt.

18. The process of claim 1, wherein said second phosphate mixture is comprised of both a sodium and a potassium salt.

19. The process of claim 1, wherein said dissolved crystal suspension from said first separation of albumin crystals is cooled to a temperature of at most 10°

C.

20. The process of claim 19, wherein the albumin crystals precipitating out of the dissolved crystal suspension are dissolved back into solution again and re-crystallized at least once.

21. The process of claim 1, wherein said albumin containing fluid is previously clarified to remove impurities not in solution.

22. The process of claim 1, wherein said feedstream is milk or other bodily fluid from a transgenic mammal.

23. The process of claim 22, wherein milk from a transgenic mammal is clarified to remove impurities and some milk proteins.

24. The process of claim 22, wherein the level of purity in a given feedstream is at least 10%, that is, wherein human albumin constitutes at least 10% of the total protein of a given solution.

25. The process according to claim 1, wherein the pH of said resultant crystallizing batch solution is 5.6-7.8.

26. The process according to claim 1, wherein the pH of said resultant crystallizing batch solution is 6.0-6.5.

27. The process according to claim 1, wherein the pH of said resultant crystallizing batch solution is 7.0-7.8.

28. The process according to claim 1, wherein said the concentration of said first phosphate mixture has a concentration in the range of 2.2-3.0 M.

29. The process of claim 1, wherein said albumin containing fluid has an albumin concentration in a range of 2-400 grams per liter of solution.

30. The process of claim 1, wherein said albumin containing fluid has an albumin concentration in a range of 3-300 grams per liter of solution.

31. The process of claim 1, wherein said albumin containing fluid has an albumin concentration in a range of 3-100 grams per liter of solution.

32. The process of claim 1, wherein said albumin containing fluid has an albumin concentration in a range of 3-40 grams per liter of solution.

33. The process of claim 1, wherein said albumin containing fluid has an albumin concentration in a range of 2-10 grams per liter of solution.

34. The process of claim 1, wherein said crystalline human albumin product is utilized as an excipient in pharmaceutical preparations.

35. The process of claim 1, wherein said crystalline human albumin product is utilized as a therapeutic agent in a pharmaceutical composition.

36. The process of claim 1, wherein said feedstream is a culture supernatant or bodily fluid derived from a host which expresses recombinant human albumin.

37. The process of claim 36, wherein said host is a mammalian cell culture.

38. The process of claim 36, wherein said host is a yeast cell culture.

39. The process of claim 36, wherein said host is an insect cell culture.

40. The process of claim 36, wherein said host is a prokaryotic cell culture.

41. The process of claim 1, wherein said crystalline human albumin product is utilized to treat a medical condition.

42. The process of claim 41, wherein said medical condition is selected from the group consisting of:

a) Edema;

b) Hypovolemia;

c) Hypoalbuminemia;

d) Adult Respiratory Distress Syndrome (ARDS);

e) Nephrosis;

f) Hemolytic Disease of the Newborn (HDN);

g) Severe burn;

h) Hypoproteinemia; and

,

i) Acute pancreatitis,

43. The process of claim 1, wherein said crystalline human albumin product is utilized during cardiopulmonary bypass surgery.

167. The process of claim 1, wherein said human albumin containing feedstream contains a compound bound to a dissolved human serum albumin product, said compound being selected from the group including:

a) Caprylate;

b) A fatty acid; and

c) A long chain alcohol.

168. The process of claim 1, wherein said human albumin product is purified to a concentrate said concentrate being in one of a variety of phases said condition being selected from the group consisting of:

a) Crystalline;

b) gel;

c) precipitant; and

d) droplet

44-86. -86. (canceled)

87. A human albumin product comprising at least a portion of the material resulting from a process of:

a) concentrating an albumin containing fluid until said fluid has at least 15 grams of albumin per liter of solution;

b) adding a first phosphate mixture to said albumin containing fluid until the concentration of said phosphate mixture is in the range of 2.6-3.0 molar and the pH is in the range of 6.1-6.3;

c) cooling the resultant filtrate of said crystallizing batch solution to a temperature of at most 15°

C.;

d) allowing the human albumin in said crystallizing batch solution to crystallize; and

e) separating the albumin crystals from any remaining fluid;

wherein the application of these specific steps allows the purification and crystallization of human albumin from a given human albumin containing feedstream.

88. The process of claim 87, wherein said first phosphate mixture is comprised of a sodium phosphate salt.

89. The process of claim 87, wherein said first phosphate mixture is a potassium phosphate salt.

90. The process of claim 87, wherein said first phosphate mixture is comprised of both a sodium and a potassium salt.

91. The process of claim 87, wherein said albumin containing fluid has a concentration of albumin in one liter of solution in a range of 15-50 grams.

92. The process of claim 87, wherein the temperature of said first phosphate mixture at the time of addition is in the range of 20-30°

C.

93. The process of claim 87, wherein said crystallizing batch solution is allowed to crystallize for at most 12 hours.

94. The process of claim 87, wherein said crystallizing batch solution is allowed to crystallize for at most 24 hours.

95. The process of claim 87, wherein said crystallizing batch solution is allowed to crystallize for at least 24 hours

96. The process of claim 87, wherein said first separation of albumin crystals is accomplished by filtration.

97. The process of claim 87, wherein said first separation of albumin crystals is accomplished by centrifugation.

98. The process of claim 87, wherein said first separation of albumin crystals is accomplished by gravity.

99. The process of claim 87, wherein said first separation of albumin crystals is accomplished by drying.

100. The process of claim 87, wherein the albumin crystals precipitating out of the dissolved crystal suspension are dissolved back into solution again and re-crystallized at least once.

101. The process of claim 87, wherein said human albumin containing fluid from said feedstream is previously clarified to remove impurities not in solution.

102. The process of claim 87, wherein said feedstream is milk or other bodily fluid from a transgenic mammal.

103. The process of claim 102, wherein milk from a transgenic mammal is clarified to remove impurities and some milk proteins.

104. The process of claim 102, wherein the level of purity in a given feedstream is at least 10%, that is, wherein human albumin constitutes at least 10% of the total protein of a given solution.

105. The process of claim 87, wherein said albumin containing fluid has an albumin concentration in a range of 2-400 grams per liter of solution.

106. The process of claim 87, wherein said albumin containing fluid has an albumin concentration in a range of 3-300 grams per liter of solution.

107. The process of claim 87, wherein said albumin containing fluid has an albumin concentration in a range of 3-100 grams per liter of solution.

108. The process of claim 87, wherein said albumin containing fluid has an albumin concentration in a range of 3-40 grams per liter of solution.

109. The process of claim 87, wherein said albumin containing fluid has an albumin concentration in a range of 2-10 grams per liter of solution.

110. The process of claim 87, wherein said crystalline human albumin product is utilized as an excipient in pharmaceutical preparations.

111. The process of claim 87, wherein said crystalline human albumin product is utilized as a therapeutic agent in a pharmaceutical composition.

112. The process of claim 87, wherein said feedstream is or is derived from a culture supernatant or bodily fluid from a host which expresses recombinant human albumin.

113. The process of claim 112, wherein said host is a mammalian cell culture.

114. The process of claim 112, wherein said host is a transgenic mammal.

115. The process of claim 112, wherein said host is a transgenic avian.

116. The process of claim 112, wherein said host is a transgenic plant.

117. The process of claim 112, wherein said host is a yeast cell culture.

118. The process of claim 112, wherein said host is an insect cell culture.

119. The process of claim 112, wherein said host is a prokaryotic cell culture.

120. The process of claim 87, wherein said crystalline human albumin product is utilized to treat a medical condition.

121. The process of claim 120, wherein said medical condition is selected from the group consisting of:

a) Edema;

b) Hypovolemia;

c) Hypoalbuminemia;

d) Adult Respiratory Distress Syndrome (ARDS);

e) Nephrosis;

f) Hemolytic Disease of the Newborn (HDN);

g) Severe burn;

h) Hypoproteinemia; and

,

i) Acute pancreatitis,

122. The process of claim 87, wherein said crystalline human albumin product is utilized during cardiopulmonary bypass surgery.

123. The process of claim 87, in which the albumin crystals are further purified by one or more recrystallization steps comprising:

a) suspending the albumin crystals from said first separation of albumin crystals in a second phosphate mixture wherein said second phosphate mixture has a concentration of 2.7-3.0 M and a pH in the range of 6.1-6.3;

b) heating the crystal suspension from said first separation of albumin crystals up to temperature in the range of 40-50°

C. in order to dissolve the crystals,

c) providing a second filtering to the dissolved crystal suspension from said first separation of albumin crystals;

d) cooling the resultant dissolved crystal suspension from said first separation of albumin crystals to a temperature of at most 15°

C.;

e) allowing albumin crystals to form from the resultant cooled crystal suspension; and

f) separating the albumin crystals from any remaining fluid;

wherein the application of these specific steps allows the purification and crystallization of human albumin from a given feedstream.

124. The process of claim 123, wherein said second phosphate mixture is comprised of a sodium phosphate salt.

125. The process of claim 123, wherein said second phosphate mixture is a potassium phosphate salt.

126. The process of claim 123, wherein said second phosphate mixture is comprised of both a sodium and a potassium salt.

143. The process of claim 102, wherein milk from a transgenic mammal is clarified to remove impurities and some milk proteins.

144. The process of claim 102, wherein the level of purity in a given feedstream is at least 10%, that is, wherein human albumin constitutes at least 10% of the total protein of a given solution.

169. The process of claim 87, wherein said human albumin containing feedstream contains a compound bound to a dissolved human serum albumin product, said compound being selected from the group including:

a) Caprylate;

b) A fatty acid; and

c) A long chain alcohol.

170. The process of claim 87, wherein said human albumin product is purified to a concentrate said concentrate being in one of a variety of phases said condition being selected from the group consisting of:

a) Crystalline;

b) gel;

c) precipitant; and

d) droplet

127. A human albumin product comprising at least a portion of the material resulting from a process of:

a) concentrating an albumin containing fluid until said fluid has at least 15 grams of albumin per liter of solution;

b) adding a first phosphate mixture to said albumin containing fluid until the concentration of said phosphate mixture is at most 2.6 molar and the pH is in the range of 6.1-6.3;

c) providing a first filtering of said albumin containing solution so as to form a resultant crystallizing batch solution to remove impurities;

d) cooling the resultant filtrate of said crystallizing batch solution to a temperature of at most 15°

C.;

e) adding more of said first phosphate mixture to said crystallizing batch solution sufficient to achieve a concentration of at most 3.0 molar;

f) allowing the human albumin in said crystallizing batch solution to crystallize; and

g) separating the albumin crystals from any remaining fluid;

wherein the application of these specific steps allows the purification and crystallization of human albumin from a given feedstream.

128. The process of claim 127, wherein said first phosphate mixture is comprised of a sodium phosphate salt.

129. The process of claim 127, wherein said first phosphate mixture is a potassium phosphate salt.

130. The process of claim 127, wherein said first phosphate mixture is comprised of both a sodium and a potassium salt.

131. The process of claim 127, wherein said albumin containing fluid has a concentration of albumin in one liter of solution in a range of 15-50 grams.

132. The process of claim 127, wherein the temperature of said first phosphate mixture at the time of addition is in the range of 20-30°

C.

133. The process of claim 127, wherein said crystallizing batch solution is allowed to crystallize for at most 12 hours.

134. The process of claim 127, wherein said crystallizing batch solution is allowed to crystallize for at most 24 hours.

135. The process of claim 127, wherein said crystallizing batch solution is allowed to crystallize for at least 24 hours.

136. The process of claim 127, wherein said first separation of albumin crystals is accomplished by filtration.

137. The process of claim 127, wherein said first separation of albumin crystals is accomplished by centrifugation.

138. The process of claim 127, wherein said first separation of albumin crystals is accomplished by gravity.

139. The process of claim 127, wherein said first separation of albumin crystals is accomplished by drying.

140. The process of claim 127, wherein the albumin crystals precipitating out of the dissolved crystal suspension are dissolved back into solution again and re-crystallized at least once.

141. The process of claim 127, wherein said human albumin containing fluid from said feedstream is previously clarified to remove impurities not in solution.

142. The process of claim 127, wherein said feedstream is milk or other bodily fluid from a transgenic mammal.

145. The process of claim 127, wherein said albumin containing fluid has an albumin concentration in a range of 2-400 grams per liter of solution.

146. The process of claim 127, wherein said albumin containing fluid has an albumin concentration in a range of 3-300 grams per liter of solution.

147. The process of claim 127, wherein said albumin containing fluid has an albumin concentration in a range of 3-100 grams per liter of solution.

148. The process of claim 127, wherein said albumin containing fluid has an albumin concentration in a range of 3-40 grams per liter of solution.

149. The process of claim 127, wherein said albumin containing fluid has an albumin concentration in a range of 2-10 grams per liter of solution.

150. The process of claim 127, wherein said crystalline human albumin product is utilized as an excipient in pharmaceutical preparations.

151. The process of claim 127, wherein said crystalline human albumin product is utilized as an therapeutic agent in a pharmaceutical composition.

152. The process of claim 127, wherein said feedstream is a culture supernatant or bodily fluid from a host which expresses recombinant human albumin.

153. The process of claim 152, wherein said host is a mammalian cell culture.

154. The process of claim 152, wherein said host is a transgenic mammal.

155. The process of claim 152, wherein said host is a transgenic avian.

156. The process of claim 152, wherein said host is a transgenic plant.

157. The process of claim 152, wherein said host is a yeast cell culture.

158. The process of claim 152, wherein said host is an insect cell culture.

159. The process of claim 152, wherein said host is a prokaryotic cell culture.

160. The process of claim 127, wherein said crystalline human albumin product is utilized to treat a medical condition.

161. The process of claim 160, wherein said medical condition is selected from the group consisting of:

a) Edema;

b) Hypovolemia;

c) Hypoalbuminemia;

d) Adult Respiratory Distress Syndrome (ARDS);

e) Nephrosis;

f) Hemolytic Disease of the Newborn (HDN);

g) Severe burn;

h) Hypoproteinemia; and

,

i) Acute pancreatitis,

162. The process of claim 127, wherein said crystalline human albumin product is utilized during cardiopulmonary bypass surgery.

163. The process of claim 127, in which the albumin crystals are further purified by one or more recrystallization steps comprising:

g) suspending the albumin crystals from said first separation of albumin crystals in a second phosphate mixture wherein said second phosphate mixture has a concentration of 2.7-3.0 M and a pH in the range of 6.1-6.3;

h) heating the crystal suspension from said first separation of albumin crystals up to temperature in the range of 40-50°

C. in order to dissolve the crystals,

i) providing a second filtering to the dissolved crystal suspension from said first separation of albumin crystals;

j) cooling the resultant dissolved crystal suspension from said first separation of albumin crystals to a temperature of at most 15°

C.;

k) allowing albumin crystals to form from the resultant cooled crystal suspension; and

l) separating the albumin crystals from any remaining fluid;

wherein the application of these specific steps allows the purification and crystallization of human albumin from a given feedstream.

164. The process of claim 163, wherein said second phosphate mixture is comprised of a sodium phosphate salt.

165. The process of claim 163, wherein said second phosphate mixture is a potassium phosphate salt.

166. The process of claim 163, wherein said second phosphate mixture is comprised of both a sodium and a potassium salt.

171. The process of claim 127, wherein said human albumin containing feedstream contains a compound bound to a dissolved human serum albumin product, said compound being selected from the group including:

a) Caprylate;

b) A fatty acid; and

c) A long chain alcohol.

172. The process of claim 127, wherein said human albumin product is purified to a concentrate said concentrate being in one of a variety of phases said condition being selected from the group consisting of:

a) Crystalline;

b) gel;

c) precipitant; and

d) droplet

173-183. -183. (canceled)