Alpha 1-antitrypsin preparation as well as a method for producing the same
A native, chromatographically purified α1-AT preparation having a purity of at least 0.7 PU/mg protein and a relative plasma α1-AT activity of at least 120% is disclosed. The ratio of active to inactive α1-AT is higher than in plasma. Furthermore, a method of producing this preparation is disclosed, as well as the use of a carrier material, e.g. an inorganic carrier material such as hydroxylapatite for separating active α1-AT from inactive α1-AT.
1. A method of preparing a purified biologically active alpha 1-antitrypsin (α1-AT) preparation containing an α1-AT isomer having a pI of between 4.3 and 4.4 comprising: providing a starting material containing active and inactive α1-AT, wherein the starting material is a plasma fraction obtained from pooled human plasma; providing a hydroxyapatite substrate; passing said starting material containing α1-AT over said hydroxyapatite substrate; eluting a biologically active α1-AT biologically active α1-AT preparation having a pI of between 4.3 and 4.4; and passing said biologically active α1-AT preparation over an anion exchange material in the presence of a detergent.
2. The method according to claim 1 , wherein said eluting step is on said hydroxyapatite is conducted with a buffer having a pH of between 5.5 and 8.0.
3. The method according to claim 2 , wherein said eluting step on hydroxyapatite is conducted with a buffer having a pH of between 6.5 and 6.8.
4. The method according to claim 1 , wherein said starting material is an albumin-depleted plasma fraction.
5. The method according to claim 1 , wherein said starting material is Cohn V precipitate.
6. The method according to claim 4 , wherein said starting material is a pre-purified α1-AT preparation fraction.
7. The method according to claim 1 , wherein said hydroxyapatite is a ceramic hydroxyapatite.
8. The method according to claim 1 , wherein said eluting step on said hydroxyapatite is conducted with a buffer which comprises a salt having an ionic strength corresponding to 60 mM of phosphate.
9. The method according to claim 1 , wherein said eluting step on said hydroxyapatite is conducted with a buffer which comprises a salt having an ionic strength corresponding to 40 mM of phosphate.
10. The method according to claim 1 , wherein said eluting step on said hydroxyapatite is conducted with a buffer which comprises a salt having an ionic strength corresponding to 50 to 130 mM of phosphate.
11. The method as set forth in claim 1 , further comprising a pathogen inactivation step.
12. The method as set forth in claim 11 , wherein said pathogen inactivation step includes at least one of a solvent, a detergent or a heat treatment step.
13. A method for purifying biologically active α1-AT including α1-AT isomers having pIs of between 4.3 and 4.4 from an alpha 1-AT-containing fraction which is obtainable from a human plasma pool comprising: Adjusting the pH of said α1-AT-containing fraction to about 6.5, absorbing said acidified α1-AT-containing fraction onto a chromatographic anion exchanger in the presence of a detergent, and eluting said biologically active α1-AT from said chromatographic anion exchanger including α1-AT isomers having pIs of between 4.3 and 4.4.
14. A method according to claim 13 in which said elution is carried out at a pH ranging between 5.5 and 8.0.
15. A method according to claim 13 in which said elution is carried out at a pH ranging between 6.5 and 6.8.