Method for preparing human serum albumin through heat-treatment in the presence of divalent cation
View Patent ↗The present invention herein discloses a method for purifying human serum albumin, which is characterized in that it comprises the steps of heat-treating a human serum albumin-containing solution including impurities originated from a host cell, in the presence of a divalent cation such as calcium ion, magnesium ion, nickel ion, cobalt ion, iron ion and zinc ion to thus selectively allow the impurities to undergo agglutination. The present invention also provides the highly purified human serum albumin prepared by the foregoing method.
1. A method for purifying human serum albumin characterized in that it comprises the steps of heat-treating at a pH value ranging from 5.5 to 10.0 a human serum albumin-containing solution including impurities originated from a host cell in the presence of a divalent cation and a stabilizer to thus allow the impurities to undergo agglutination and then removing aggregates thus generated from the human serum albumin-containing solution.
2. A method for producing human serum albumin starting from a raw material obtained through gene engineering operations characterized in that it comprises the steps of heat-treating at a pH value ranging from 5.5 to 10.0 a human serum albumin-containing solution including impurities originated from a host cell in the presence of a divalent cation and a stabilizer to thus allow the impurities to undergo agglutination and then removing aggregates thus generated from the human serum albumin solution.
3. The method as set forth in claim 1 wherein the human serum albumin-containing solution has, before the heat-treating, a human serum albumin concentration ranging from 0.01 to 30%.
4. The method as set forth in claim 1 wherein the human serum albumin-containing solution has, before heat-treating, a human serum albumin concentration ranging from 0.1 to 10%.
5. The method as set forth in claim 1 wherein the divalent cation is selected from the group consisting of calcium ion, magnesium ion, nickel ion, cobalt ion, iron ion and zinc ion.
6. The method as set forth in claim 1 wherein the concentration of the divalent cation ranges from 1 to 1000 mM.
7. The method as set forth in claim 1 wherein the concentration of the divalent cation ranges from 100 to 500 mM.
8. The method as set forth in claim 1 wherein the stabilizer is acetyl tryptophan or a salt thereof and/or a fatty acid having 6 to 20 carbon atoms or a salt thereof.
9. The method as set forth in claim 8 wherein the fatty acid salt is sodium caprylate.
10. The method as set forth in claim 9 wherein the concentration of the sodium caprylate ranges from 5 to 20 mM.
11. The method as set forth in claim 1 wherein the heat-treatment is carried out at a temperature ranging from 50 to 95° C.
12. The method as set forth in claim 1 wherein the heat-treatment is carried out at a temperature ranging from 60 to 75° C.
13. The method as set forth in claim 1 wherein the heat-treatment is carried out for a time ranging from one minute to 30 hours.
14. The method as set forth in claim 1 wherein the heat-treatment is carried out for a time ranging from 1 to 5 hours.
15. The method as set forth in claim 1 wherein the heat-treatment is carried out at a pH value ranging from 9 to 10.
16. The method as set forth in claim 1 wherein the step for the removal of the aggregates includes the use of a low speed centrifugation technique, an ultrafiltration technique whose fractional molecular weight ranges from 100,000 to 300,000, or the combination of these techniques.
17. A method for preparing human serum albumin starting from a raw material obtained through gene engineering operations characterized in that it comprises the following steps:
(1) heat-treating a human serum albumin-containing solution including impurities originated from a host cell and having a human serum albumin concentration ranging, before heat-treating, from 5 to 10%, in the presence of calcium ion in an amount ranging from 100 to 500 mM, while optionally adding sodium caprylate in an amount ranging from 5 to 20 mM, at a pH value ranging from 9 to 10 and a temperature ranging from 60 to 75° C. for a time ranging from 1 to 5 hours to thus allow the impurities to undergo agglutination; and
(2) removing aggregates thus generated from the human serum albumin-containing solution using a low speed centrifugation technique, an ultrafiltration technique whose fractional molecular weight ranges from 100,000 to 300,000, or the combination of these techniques.
18. The method as set forth in claim 2 wherein the human serum albumin-containing solution has, before the heat-treating, a human serum albumin concentration ranging from 0.01 to 30%.
19. The method as set forth in claim 2 wherein the human serum albumin-containing solution has, before heat-treating, a human serum albumin concentration ranging from 0.1 to 10%.
20. The method as set forth in claim 2 wherein the divalent cation is selected from the group consisting of calcium ion, magnesium ion, nickel ion, cobalt ion, iron ion and zinc ion.
21. The method as set forth in claim 2 wherein the concentration of the divalent cation ranges from 1 to 1000 mM.
22. The method as set forth in claim 2 wherein the concentration of the divalent cation ranges from 100 to 500 mM.
23. The method as set forth in claim 2 wherein the stabilizer is acetyl tryptophan or a salt thereof and/or a fatty acid having 6 to 20 carbon atoms or a salt thereof.
24. The method as set forth in claim 23 wherein the fatty acid salt is sodium caprylate.
25. The method as set forth in claim 24 wherein the concentration of the sodium caprylate ranges from 5 to 20 mM.
26. The method as set forth in claim 2 wherein the heat-treatment is carried out at a temperature ranging from 50 to 95° C.
27. The method as set forth in claim 2 wherein the heat-treatment is carried out at a temperature ranging from 60 to 75° C.
28. The method as set forth in claim 2 wherein the heat-treatment is carried out for a time ranging from one minute to 30 hours.
29. The method as set forth in claim 2 wherein the heat-treatment is carried out for a time ranging from 1 to 5 hours.
30. The method as set forth in claim 2 wherein the heat-treatment is carried out at a pH value ranging from 9 to 10.
31. The method as set forth in claim 2 wherein the step for the removal of the aggregates includes the use of a low speed centrifugation technique, an ultrafiltration technique whose fractional molecular weight ranges from 100,000 to 300,000, or the combination of these techniques.
32. A method for purifying human serum albumin characterized in that it consists of the steps of heat-treating at a pH value ranging from 5.5 to 10.0 a human serum albumin-containing solution including impurities originated from a host cell in the presence of a divalent cation and a stabilizer to thus allow the impurities to undergo agglutination and then removing aggregates thus generated from the human serum albumin-containing solution.
33. The method as set forth in claim 32 wherein the heat-treatment is carried out at a pH value ranging from 9 to 10.
34. A method for producing human serum albumin starting from a raw material obtained through gene engineering operations characterized in that it consists of the steps of heat-treating at a pH value ranging from 5.5 to 10.0 a human serum albumin-containing solution including impurities originated from a host cell in the presence of a divalent cation and a stabilizer to thus allow the impurities to undergo agglutination and then removing aggregates thus generated from the human serum albumin solution.
35. The method as set forth in claim 34 wherein the heat-treatment is carried out at a pH value ranging from 9 to 10.