Method for the purification of alpha-1 proteinase inhibitor (a1PI)
View Patent ↗The present invention relates to a method for the purification of alpha-1 proteinase inhibitor (a1PI) from protein fractions. More specifically, the invention relates to an improved method for the purification of alpha-1 proteinase inhibitor (a1PI), wherein the yield of a1PI can be increased by thawing the starting material and incubating it for several hours before subjecting it to a washing step.
1. A method for the purification of alpha-1 proteinase inhibitor (a1PI) from a protein precipitate, comprising the steps of
a. providing a frozen protein precipitate comprising a1PI, wherein the frozen protein precipitate comprises at least one Cohn fraction selected from the group consisting of Cohn fraction IV 1 , Cohn fraction IV 4 , Cohn fraction IV 1 +IV 4 and Cohn fraction IV 1,4 ,
b. thawing the protein precipitate at an ambient temperature of 2-25° C. until the protein precipitate has reached ambient temperature, without adding a suspending solvent,
c. maintaining the thawed protein precipitate for at least 15 h (hours) at an ambient temperature of 2-25° C., and
d. suspending the thawed, incubated precipitate of step c in a wash solution, and washing the protein precipitate; and
e. extracting and purifying a1PI from the thawed, washed protein precipitate of step d.
2. A method according to claim 1 , wherein the Cohn fraction is obtained by using filtration as separation method and wherein the Cohn fraction comprises a filter aid.
3. A method according to claim 2 , wherein the filter aid comprises 96-98% SiO 2 .
4. A method according to claim 1 , wherein the frozen protein precipitate comprising a1PI is derived from a1PI produced by recombinant methods.
5. A method according to claim 1 , wherein the frozen protein precipitate is of human origin.
6. A method according to claim 1 , wherein the frozen protein precipitate is crushed.
7. A method according to claim 1 , wherein the frozen protein precipitate is thawed at an ambient temperature of about 4° C.
8. A method according to claim 1 , wherein the thawed protein precipitate is incubated at an ambient temperature of about 4° C.
9. A method according to claim 1 , wherein the thawed protein precipitate is incubated for 15 h.
10. A method according to claim 1 , wherein the thawed protein precipitate is incubated for 16 h to 19 h.
11. A method according to claim 1 , wherein the suspending and washing step d comprises suspending the thawed and incubated protein precipitate in water or saline solution at a temperature of less than about 15° C. for at least 1 h, subsequently adjusting the pH of the washing suspension to about 5.5 or 6.0, and separating the soluble proteins from the insoluble proteins by filtration or centrifugation.
12. A method according to claim 11 , wherein the soluble proteins are separated from the insoluble proteins by filtration and a filter aid is used for said separation.
13. A method according to claim 12 , wherein the filter aid comprises 96-98% SiO 2 .
14. A method according to claim 1 , wherein the washing step is conducted at least twice.