Purification of human serum albumin

• US 20030036637A1
• Filed: 06/13/2002
• Published: 02/20/2003
• Est. Priority Date: 06/13/2001
• Status: Abandoned Application

• Current Assignee: Taurus HSA LLC
• Original Assignee: Taurus HSA LLC
• Inventors: Fulton, Scott
• Application Number:
  US10/172,159
• Publication Number:
  US 20030036637A1
• Time in Patent Office: Days
• US Class Current: 530/363
• 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: 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: C07K 14/765 Serum albumin, e.g. HSA

Abstract

The invention features methods of purifying human serum albumin (hSA) from endogenous serum albumin of the host cell producing the hSA. The methods include providing a sample comprising hSA and serum albumin of the host cell, applying the sample to an affinity column that binds hSA at a higher affinity than the serum albumin of the host cell, eluting bound hSA from the affinity column, and crystallizing the eluted has. The invention also features compositions comprising hSA produced by the methods of the invention.

Claims

1. A method of purifying human serum albumin (hSA) from a sample that contains hSA and serum albumin of a host cell comprising:

obtaining a sample from a host cell that contains hSA and serum albumin of a host cell;

applying the sample to an affinity column that binds hSA at a higher affinity than the serum albumin of the host cell;

eluting bound hSA from the affinity column; and

crystallizing the eluted hSA.

2. The method of claim 1, wherein the sample is obtained from a transgenic non-human animal.

3. The method of claim 2, wherein the animal is selected from the group consisting of a cow, a sheep, a goat, a pig, a mouse and a rabbit.

4. The method of claim 2, wherein the sample is obtained from the milk, blood, or tissue of the mammal.

5. The method of claim 1, wherein the sample is medium that has been used to culture cells.

6. The method of claim 2, wherein the sample is obtained from the milk of a transgenic mammal which produces hSA in its mammary epithelial cells.

7. The method of claim 6, wherein the method further comprises decreaming the milk sample.

8. The method of claim 6, wherein the method further comprises treating the milk sample to remove casein.

9. The method of claim 8, wherein the casein is removed by acid precipitation, centrifugation or tangential flow filtration.

10. The method of claim 6, wherein the sample is a clarified milk sample.

11. The method of claim 10, wherein the clarified milk sample is in a salt buffer.

12. The method of claim 11, wherein the salt buffer comprises 250 mM NaCl at pH 8.5 and a low concentration of a non-ionic detergent.

13. The method of claim 11, wherein the affinity column comprises a synthetic ligand resin.

14. The method of claim 13, wherein the synthetic ligand resin uses a dye Reactive Blue 2 or a modified dye Reactive Blue 2 as an affinity ligand.

15. The method of claim 1, wherein the affinity column does not substantially bind to the serum albumin of the host cell as compared to its affinity to bind hSA.

16. The method of claim 1, further comprising washing the affinity column after the sample has been applied to the column.

17. The method of claim 16, wherein the wash buffer comprises 250 mM NaCl at pH 8.5 and a low concentration of a non-ionic detergent.

18. The method of claim 1, wherein the hSA is eluted from the affinity column using an elution buffer does not substantially induce the elution of non-serum albumin proteins bound to the affinity column.

19. The method of claim 18, wherein the elution buffer comprises a phosphate buffer and a fatty acid molecule that competes with the affinity ligand of the column for binding to hSA.

20. The method of claim 19, wherein the elution buffer comprises about 20-50 mM phosphate at about pH 6.0.

21. The method of claim 19, wherein the fatty acid molecule is caprylate.

22. The method of claim 1, further comprising applying the affinity-purified hSA sample to the affinity column or a second affinity column, washing the hSA bound affinity column and eluting the hSA bound to the affinity column to thereby produce a twice affinity-purified hSA sample.

23. The method of claim 1, wherein the bound hSA is crystallized by adding a crystallizing agent to the sample.

24. The method of claim 23, wherein the crystallizing agent is selected from the group consisting of polyethylene glycol (PEG), ammonium sulfate, phosphate, or combinations thereof.

25. The method of claim 23, wherein the crystallizing agent is phosphate and is added to a final concentration of 2.7 to 2.8 M.

26. The method of claim 23, wherein the crystallizing agent further comprises a fatty acid molecule that binds to hSA.

28. The method of claim 1, wherein the crystallized hSA is separated from the crystallization solution.

29. A composition comprising hSA made by the method of claim 1.

27. The method of claim 27, wherein the fatty acid molecule is caprylate.