Process for purifying a compound

• US 20040225120A1
• Filed: 06/08/2004
• Published: 11/11/2004
• Est. Priority Date: 09/11/1996
• Status: Active Grant

• Current Assignee: Pfizer Inc.
• Original Assignee: Folke Tjernelo, Hans Ageland, Josefine Persson, Lena Nystrom
• Inventors: Tjernelo, Folke, Ageland, Hans, Persson, Josefine, Nystrom, Lena
• Granted Patent: US 7,193,056 B2
• Time in Patent Office: Days
• US Class Current: 536/124
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/775 Apolipopeptides

Abstract

The present invention relates to a process for purifying a hydrophobic or amphiphilic compound, by first mixing a starting material containing the hydrophobic or amphiphilic compound with a first polymeric material, water and at least one of a second polymeric material and a surfactant, wherein the first polymeric material and the second polymeric material and/or surfactant are immiscible in the resulting primary aqueous solution. The process further comprises maintaining the primary aqueous solution for a period of time sufficient for essentially separating the phases formed, and then removing the phase containing the main portion of the hydrophobic or amphiphilic compound and the second polymeric material and/or surfactant. The second polymeric material and/or surfactant are separated from the hydrophobic or amphiphilic compound, and subsequently recycled to the initial mixing step. The present invention further relates to a composition for use in purification of apolipoprotein A (ApoA) or apolipoprotein E (ApoE), said composition comprising a first polymeric material and a surfactant, said first polymeric material and surfactant being immiscible in the primary aqueous solution obtained after mixing with water. ApoA or ApoE produced by the inventive process can be used for the manufacture of a medicament in the treatment of atherosclerosis and cardiovascular diseases, sepsis or peripheral atherosclerosis as well as in a method for treatment of atherosclerosis and cardiovascular diseases, sepsis or peripheral atherosclerosis when administered in a therapeutically effective amount.

Claims

1-36. -36. (Cancelled).

37. A process for purifying recombinantly produced apolipoprotein ApoA-I Milano, comprising

a) mixing, in arbitrary order, a starting material containing the apolipoprotein, a first hydrophilic polymeric material, water, a second amphiphilic polymeric material and a surfactant, the first polymeric material and the second polymeric material being immiscible in the resulting primary aqueous solution;

b) maintaining the primary aqueous solution for a period of time sufficient for essentially separating the solution into phases;

c) removing a phase containing a main portion of the apolipoprotein and the second polymeric material;

d) separating the apolipoprotein from the second polymeric material; and

subsequently

e) recycling the separated second polymeric material to the initial mixing step (a).

38. A process according to claim 37, wherein the first polymeric material, the second polymeric material and the surfactant are mixed with water and the starting material containing the apolipoprotein.

39. A process according to claim 37, wherein the first polymeric material is selected from the group consisting of hydroxyalkyl cellulose, hydroxyalkyl starches, starch, dextran, pullulan, and derivatives and mixtures thereof.

40. A process according to claim 39, wherein the hydroxyalkyl starch is selected from the group consisting of hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, and mixtures thereof.

41. A process according to claim 37, wherein the molecular weight of the first polymeric material lies in the range of from about 5,000 up to about 5,000,000 Da.

42. A process according to claim 37, wherein the second polymeric material exhibits inverse solubility characteristics.

43. A process according to claim 42, wherein the second polymeric material has a cloud point in the range of from about 5°

C. up to about 90°

C.

44. A process according to claim 37, wherein the second polymeric material is selected from the group consisting of polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methylether, alkoxylated starches, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and derivatives and mixtures thereof.

45. A process according to claim 37, wherein the poly(oxyalkylene) copolymer is a random or blocked copolymer of ethylene oxide and propylene oxide.

46. A process according to claim 45, wherein the content of propylene oxide of the copolymer lies in the range of from about 30 to about 90% by weight of the total weight of the copolymer.

47. A process according to claim 42, wherein the apolipoprotein is separated from the second polymeric material by heating the removed phase to a temperature below the temperature where degradation of the apolipoprotein occurs but above the cloud point of the second polymeric material for precipitating the second polymeric material, and subsequently separating a phase containing the precipitated second polymeric material from a phase containing the apolipoprotein.

48. A process according to claim 37, wherein the concentration of the first polymeric material in the primary aqueous solution lies in the range of from about 1% to about 30% by weight of the total weight of the primary aqueous solution.

49. A process according to claim 37, wherein the concentration of the second polymeric material in the primary aqueous solution lies in the range of from about 0.5% to about 30% by weight of the total weight of the primary aqueous solution.

50. A process according to claim 37, wherein the concentration of the apolipoprotein in the primary aqueous solution lies in the range of from about 0.1 g/l to about 50 g/l of the primary aqueous solution.

51. A process according to claim 37, wherein the surfactant is selected from the group consisting of non-ionic surfactants, anionic surfactants and cationic surfactants.

52. A process according to claim 51, wherein the non-ionic surfactant is selected from the group consisting of alkyl ethoxylates, polyoxyethylene sorbitan fatty esters, block copolymers, polyoxyethylene alkyl ethers, polyglycol ethers and alkyl glycosides.

53. A process according to claim 37, wherein the concentration of the surfactant in the primary aqueous solution is above the critical micelle concentration (CMC).

54. A process according to claim 37, wherein at least about 60% of the initial amount of the second polymeric material is recycled to the initial mixing step.

55. A process according to claim 37, wherein the starting material is a fermentation broth.

56. A process according to claim 41, wherein the molecular weight of the first polymeric material lies in the range of from 40,000 to 500,000 Da.

57. A process according to claim 43, wherein the second polymeric material has a cloud point in the range of from 15°

C. to 60°

C.

58. A process according to claim 46, wherein the content of propylene oxide of the copolymer lies in the range of from 40% to 80% by weight of the total weight of the copolymer.

59. A process according to claim 48, wherein the concentration of the first polymeric material in the primary aqueous solution lies in the range of from 3% to 20% by weight.

60. A process according to claim 49, wherein the concentration of the second polymeric material in the primary aqueous solution lies in the range of from 3% to 20% by weight of the total weight of the primary aqueous solution.

61. A process according to claim 50, wherein the concentration of the apolipoprotein in the primary aqueous solution lies in the range of from 1 g/l to 10 g/l of the primary aqueous solution.

62. A process according to claim 54, wherein at least 80% of the initial amount of the component is recycled to the initial mixing step.

63. A process according to claim 37, wherein the surfactant is amphiphilic.

64. A process according to claim 37, wherein the primary aqueous solution essentially separates into two phases.

65. A process for purifying recombinantly produced apolipoprotein ApoA-I Milano, comprising

a) mixing, in arbitrary order, a starting material containing the apolipoprotein, a first hydrophilic polymeric material, water, a second amphiphilic polymeric material and a surfactant, the first polymeric material and the second polymeric material being immiscible in the resulting primary aqueous solution;

b) maintaining the primary aqueous solution for a period of time sufficient for essentially separating the solution into phases;

c) removing a phase containing a main portion of the apolipoprotein, the second polymeric material and the surfactant;

d) separating the apolipoprotein from the second polymeric material and the surfactant; and

subsequently

e) recycling the second polymeric material and the surfactant to the initial mixing step (a).

66. A process according to claim 65, wherein the first polymeric material is selected from the group consisting of hydroxyalkyl cellulose, hydroxyalkyl starches, starch, dextran, pullulan, and derivatives and mixtures thereof.

67. A process according to claim 66, wherein the hydroxyalkyl starch is selected from the group consisting of hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, and mixtures thereof.

68. A process according to claim 65, wherein the second polymeric material exhibits inverse solubility characteristics.

69. A process according to claim 68, wherein the second polymeric material has a cloud point in the range of from about 5°

C. to about 90°

C.

70. A process according to claim 65, wherein the second polymeric material is selected from the group consisting of polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methylether, alkoxylated starches, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and derivatives and mixtures thereof.

71. A process according to claim 70, wherein the poly(oxyalkylene) copolymer is a random or block copolymer of ethylene oxide and propylene oxide.

72. A process according to claim 65, wherein the primary aqueous solution essentially separates into two phases.

73. A process for purifying recombinantly produced apolipoprotein ApoA-I Milano, comprising

a) mixing, in arbitrary order, a starting material containing the apolipoprotein, a first hydrophilic polymeric material, water and a surfactant, the first polymeric material and the surfactant being immiscible in the resulting primary aqueous solution;

b) maintaining the primary aqueous solution for a period of time sufficient for essentially separating the solution into two phases;

c) removing a phase containing a main portion of the apolipoprotein and the surfactant;

d) separating the apolipoprotein from the surfactant; and

subsequently e) recycling the surfactant to the initial mixing step (a).