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
1991-12-04
1994-04-19
Wityshyn, Michael G.
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...
530364, 530416, 530417, 210263, 210635, 210656, 210660, 210661, 210666, 210683, 210685, 210702, 210724, 210734, 210736, A61K 3514, B01D 1508
Patent
active
053046382
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to improved media for the separation of blood coagulation factors from plasma concentrates and methods of separation and isolation using these media.
DESCRIPTION OF THE RELATED ART
Blood Coagulation Factor VIII (abbreviated herein to factor VIII) is a protein component of blood, the absence or deficiency of which results in haemophilia. It is obtained from donated blood plasma in many countries on an industrial scale for administration to haemophiliacs. Factor VIII is present in plasma at very low concentrations and current processes attempt to produce purified factor VIII-containing products in which the specific activity of the factor VIII has been significantly increased relative to other plasma proteins. In general factor VIII is isolated in the form of a complex, the complex being formed with von Willebrand Factor (vWF).
One method of purification of factor VIII which has been employed is adsorption onto and elution from a separation medium having an affinity for factor VIII. In that the affinity of the medium for other proteins is normally different from that for factor VIII, some separation may be achieved, particularly using chromatographic techniques.
It is important in such separation techniques that the separation medium not only shows some selectivity in the adsorption of factor VIII but also that large amounts of factor VIII can be adsorbed per unit volume of separation medium and can be eluted at a high rate of recovery Hitherto, such separation media for factor VIII have consisted of an inert substrate such as an agarose, e.g. Sepharose, carrying functional groupings having an affinity for factor VIII, notably aminoalkyl groups. Typical materials of this type have been described by Austen DEG (1979: Br. J.Haematol. 43, 669-674); Austen DEG and Smith JK (1982: Thromb.Haemostas. 48, 46-48) and U.S. Pat. No. 4,508,709.
In particular, Neal G.G. et al have described in a Poster presentation in San Diego in July 1985 certain separation media for factor VIII comprising Sepharose bound to a wide range of amines including certain polyamines. (Neal GG, Smith JK and Hersee D, 1985, Thromb.Haemostas. 54, 78 Conference Abstract). The polyamines showed better absorption of factor VIII than the simple monoamines but alkylenediamines gave better results than dialkylenetriamines such as diethylenetriamine or di(n-propylene)-triamine .
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a graph illustrating the linear relationship between Q value and sodium chloride concentration required to elute Factor VIII from various polyamine ligands attached to sepharose.
Our further investigations have revealed that higher polyamine groups are capable of giving better results in isolation of factor VIII than the amine groups described by Neal et al, and that furthermore, a high number of protonated amino groups relative to the number of methylene groups is important in determining the selectivity for factor VIII. We have found, in this context, that the polyamine having the formula H.sub.2 N(CH.sub.2).sub.2 NH(CH.sub.2).sub.3 NH.sub.2) (N-(2-aminoethyl)-1,3-propanediamine) gave better results than either H.sub.2 N(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 NH.sub.2 (diethylenetriamine) or H.sub.2 N(CH.sub.2).sub.3 NH(CH.sub.2).sub.3 NH.sub.2 (di(n-propylene) triamine), and polyamines having four or more nitrogen atoms, such as triethylenetetramine (TETA), gave even better results. In the case of diethylenetriamine, the proximity of the nitrogens prevents full protonation of all three at appropriate pH levels e.g. pH 5.5, whereas the introduction of one further methylene group, to give the compound H.sub.2 N(CH.sub.2).sub.2 NH(CH.sub.2).sub.3 NH.sub.2 allows full protonation at pH 5.5.
According to the present invention therefore we provide a separation medium for use in protein separation comprising a water-insoluble matrix carrying a plurality of polyamine groupings, said polyamine groupings having at least 3 basic nitrogen atoms, said basic nitrogen
REFERENCES:
patent: 3860573 (1975-01-01), Honkanen et al.
patent: 4157431 (1979-06-01), Fields et al.
patent: 4397841 (1983-08-01), Johnson
patent: 4471112 (1984-09-01), Johnson
patent: 4508709 (1985-04-01), Amphlett
patent: 4675384 (1987-06-01), Dromard et al.
patent: 4883598 (1989-11-01), Riethorst et al.
Clark, Jr. et al, Experimental Biochemistry, second edition, W. H. Freeman and Company, San Francisco, 1977, pp. 15-20.
Morgenthaler, Thromb. Haemostas., 47:124-127 (1982).
Neal et al, Thromb. Haemostas., 54:78 (1985).
Tuddenham et al, British Journal of Haematology, 52:259-267 (1982).
Austen, British Journal of Haematology, 43:669-674 (1979).
Austen et al, Thromb. Haemostas., 48:46-48 (1982).
Lowe Christopher R.
Marshall Philip J.
Central Blood Laboratories Authority
Mohamed Abdel A.
Wityshyn Michael G.
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