THROMBIN PURIFICATION
The invention relates to thrombin compositions with reduced levels of high molecular weight impurities. In particular, the levels of factor Va, prions and/or viral agents are greatly reduced. This invention also relates generally to methods for the preparation of thrombin having a high degree of purity and high specific activity. More specifically, the invention encompasses steps to exclude high molecular weight impurities from thrombin preparations by size exclusion filtration. In additional embodiments, the preparation of thrombin additionally includes an ion exchange filtration step. The methods of the invention are particularly suited for large scale purification of thrombin. This invention also relates generally to stabilized formulations containing thrombin compositions. More specifically, the present invention relates to stabilized, liquid formulations containing thrombin having a high degree of purity and high specific activity and methods of making and using such formulations.
1 . A process for the preparation of a purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa, and having a specific activity greater than 1800 units of thrombin per mg (u/mg) of protein, which process comprises:
(a) applying a thrombin preparation having from about 500 to about 3000 units of thrombin per mL (u/mL) of the preparation to a cation exchange chromatography to afford a pre-purified thrombin composition having a specific activity greater than about 1500 u/mg of protein;
(b) applying the pre-purified thrombin composition from step (a) to a size exclusion filtration capable of excluding impurities having a molecular weight greater than 40 kDa to afford a purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa, and having a specific activity greater than 1800 u/mg of protein; and optionally,
(c) applying the purified thrombin composition from step (b) to an anion exchange filtration.
2 . The process of claim 1 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight greater than 50 kDa.
3 . The process of claim 1 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight greater than 100 kDa.
4 . The process of claim 1 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight ranging from 40 kDa to 300 kDa.
5 . The process of claim 1 , wherein the purified thrombin composition has a specific activity between about 1800 and about 3000 u/mg of protein.
6 . The process of claim 1 , wherein the purified thrombin composition has a specific activity between about 2300 and about 2700 u/mg of protein.
7 . The process of claim 1 , wherein the purified thrombin composition is substantially free of factor Va.
8 . The process of claim 7 , wherein the factor Va is present at less than 0.4 μg per 1000 units of thrombin.
9 . The process of claim 1 , wherein the purified thrombin composition is substantially free of prions.
10 . The process of claim 1 , wherein the purified thrombin composition is substantially free of viral agents.
11 . The process of claim 10 , wherein the viral agent is selected from the group consisting of bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV), bovine parvovirus (BPV), canine parvovirus (CPV), stickleback virus (SBV), tick-borne encephalitis virus (TBEV), equine rhinovirus 1 (ERV-1), human immunodeficiency virus 1 (HIV-1), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and xenotropic murine leukemia virus (XMuLV).
12 . The process of claim 1 , wherein the purified thrombin composition has a viral clearance log reduction value of at least about 3.5 logs.
13 . A purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa, obtainable by the process comprising:
(a) applying a thrombin preparation to a cation exchange chromatography to afford a pre-purified thrombin composition;
(b) applying the pre-purified thrombin composition from step (a) to a size exclusion filtration capable of excluding impurities having a molecular weight greater than 40 kDa to afford a purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa; and optionally,
(c) applying the purified thrombin composition from step (b) to an anion exchange filtration.
14 . A purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa, and having a specific activity greater than 1800 units of thrombin per mg (u/mg) of protein obtainable by the process comprising:
(a) applying a thrombin preparation having from about 500 to about 3000 units of thrombin per mL (u/mL) of the preparation to a cation exchange chromatography to afford a pre-purified thrombin composition having a specific activity greater than about 1500 u/mg of protein;
(b) applying the pre-purified thrombin composition from step (a) to a size exclusion filtration capable of excluding impurities having a molecular weight greater than 40 kDa to afford a purified thrombin composition, substantially free of impurities having a molecular weight greater than 40 kDa, and having a specific activity greater than 1800 u/mg of protein; and optionally,
(c) applying the purified thrombin composition from step (b) to an anion exchange filtration.
15 . The purified thrombin composition of claim 14 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight greater than 50 kDa.
16 . The purified thrombin composition of claim 14 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight greater than 100 kDa.
17 . The purified thrombin composition of claim 14 , wherein the size exclusion filtration is capable of excluding impurities having a molecular weight ranging from 40 kDa to 300 kDa.
18 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition has a specific activity between about 1800 and about 3000 u/mg of protein.
19 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition has a specific activity between about 2300 and about 2700 u/mg of protein.
20 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition is substantially free of factor Va.
21 . The purified thrombin composition of claim 20 , wherein the factor Va is present at less than 0.4 μg per 1000 units of thrombin.
22 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition is substantially free of prions.
23 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition is substantially free of viral agents.
24 . The purified thrombin composition of claim 23 , wherein the viral agent is selected from the group consisting of bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV), bovine parvovirus (BPV), canine parvovirus (CPV), stickleback virus (SBV), tick-borne encephalitis virus (TBEV), equine rhinovirus 1 (ERV-1), human immunodeficiency virus 1 (HIV-1), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and xenotropic murine leukemia virus (XMuLV).
25 . The purified thrombin composition of claim 14 , wherein the purified thrombin composition has a viral clearance log reduction value of at least about 3.5 logs.