Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1997-11-26
1999-07-06
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530381, 530382, 530383, 530412, 530413, 530414, 530416, 530418, 530422, 530427, 435236, 435238, 435239, 424529, 424532, A61K 3514, C12N 704
Patent
active
059199091
DESCRIPTION:
BRIEF SUMMARY
SUMMARY OF THE INVENTION
The present invention pertains to a process for the preparation of factor IX from biological sources by chromatography as well as to factor IX obtainable by the process according to the invention.
When factor IX is prepared, for example, from blood plasma, the cryoprecipitate is first separated. The frozen citrate plasma is thawed and the major amount of factor VIII and fibrinogen is separated by centrifugation. The cryopoor plasma thus obtained contains the vitamin K dependent coagulation factors with an activity in the order of magnitude of 0.01 IU/mg of protein.
According to the process known from the prior art, the vitamin K dependent coagulation factors are bound by extraction with anion exchangers, such as DEAE Sephadex, without adversely affecting albumin recovery. However, due to the poor flow characteristics of DEAE Sephadex, this step must usually be performed as a solid phase extraction in batch operation. The fractions obtained after elution of the DEAE Sephadex then serve as the starting material for the purification of factor IX. Proteases contained in the plasma, however, are also enriched in this fraction, resulting in yield losses of the material of interest, factor IX, by proteolytic digestion and an increase of the risk of thrombogenity of the preparation.
The object of the invention is to provide a process which reduces the risks mentioned without the addition of protease inhibitors.
Surprisingly, this object is achieved by a process with the features of the claim. This involves treating the biological source containing factor IX with a protein precipitant. Candidate biological sources are, in particular, blood plasma or cryoprecipitate depleted of factor VIII and fibrinogen (cryopoor plasma).
The concentration of the protein precipitant is, in particular, from 1.5 to 2.3 mol/l, preferably from 1.75 to 1.9 mol/l. The values relate to the protein precipitant concentrations present in the sources containing factor IX (final concentration).
As the protein precipitant, ammonium sulfate is considered in particular.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a flow chart illustrating chromatographic purification according to the instant invention.
The process according to the invention reduces the proteolytic activity by at least one order of magnitude. In addition, interfering components, such as factor VII and factor II, are removed by the precipitation step prior to the actual chromatographic purification of factor IX. Proteins C and S are also depleted. In the case of factor VII, almost complete elimination can be achieved. Factor II is also depleted rather completely (70 to 90%) whereas depletion of proteins C and S is about 50%.
In the supernatant of the fraction treated with the protein precipitant, the activity of factor IX is typically from 20 to 30 IU/ml, corresponding to a specific activity of 1 to 5 IU/mg.
The precipitation step according to the invention, preferably using ammonium sulfate, is followed by the chromatographic separation of the fraction enriched in factor IX. This takes advantage of the fact that at high salt concentrations proteins can undergo hydrophobic interactions to different extents. According to the invention, factor IX is preferably bound in the presence of relatively high salt concentrations, which are present from protein precipitation, to a chromatographic material capable of being employed for hydrophobic interaction chromatography (HIC). This at the same time reduces the high salt concentration resulting from protein precipitation. In particular, a chromatographic material may be used which has bound on the substrate material a hydrophilic flexible arm (tentacle). Covalently bound to these spacers are then the corresponding ligands, in particular hydrophobic ligands, such as propyl, butyl, phenyl groups.
The supernatant of the fraction treated with the protein precipitant is applied to the appropriate chromatographic material and optionally washed. Elution of the factor IX bound to the column and still containing factor X c
REFERENCES:
patent: 4687664 (1987-08-01), Philopitsch et al.
patent: 5281661 (1994-01-01), Linnau et al.
patent: 5457181 (1995-10-01), Michalski et al.
"HiLoad.TM. Phenyl Sepharose.RTM. High Performance," Data File, Pharmacia Biotech. (18-1022-55).
Stampe et al., Journal of Chromatography, vol. 363, pp. 101-103, 1986.
R. K. Scopes, Protein Purification: Principles and Practice (Second Edition), 1987.
Hoffer Lutz
Josic Djuro
Morfeld Frank
Mohamed Abdel A.
Octapharma AG
Tsang Cecilia J.
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