Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-08-01
2004-11-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
Reexamination Certificate
active
06815535
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates primarily to a method of obtaining fibrinogen. The method of the present invention also enables the obtaining of fibronectin and Factor XIII.
BACKGROUND OF THE INVENTION
The isolation of human fibrinogen has traditionally been carried out by classical plasma fractionation methods. Fibrinogen is precipitated from plasma either with ethanol (Blomback and Blomback, 1956), ammonium sulphate (Takeda, 1996), &bgr; alanine/glycine (Jakobsen and Kieruif, 1976), polymers (polyethelene glycol) and low ionic strength solutions (Holm, 1985) with relative high yield and homogeneity.
Further purification of fibrinogen precipitates can be achieved by ion-exchange chromatography conditions (Stathakis, 1978) and affinity chromatography (Kuyas, 1990). Specific contaminants can be absorbed out for example fibronectin an immobilised gelatine and plasminogen an immobilised lysine (Vuento, 1979).
Over the past few decades the overall structure and function of the fibrinogen molecule has been elucidated. The completion of the amino acid sequence of human fibrinogen (Henschen and Lottspeich, 1977) and the assignment of disulphide bonds (Blomback et al. 1976, Douma et al. 1978) provided data that confirmed the pioneer observations of an extended multidomained molecule (Hah and Slayter, 1959). The cloning of the fibrinogen genes and the complete amino acid sequence of all three chains of human fibrinogen from the cDNA studies are in agreement with those reported earlier based on the conventional amino acid sequencing procedures (Chung et al. 1983).
Precipitation methods are widely used for the manufacture of commercial fibrinogen. Chromatographic methods are now being explored as an alternative or to improve the purity of fibrinogen concentrates.
Fibrinogen interacts with a number of physiologically important proteins (Doolittle, 1984) such a plasminogen, thrombin, fibronectin, certain strains of staphylococcal bacteria and platelets. A number of functional characteristics have been assigned to specific parts of the molecule including: the position of the fibrinopeptides released from the parent molecule by catalytic action of thrombin, fibrin covalent stabilisation donor and acceptor sites, carbohydrate clusters, polymerisation sites, calcium binding sites and the attachment sites for fibronectin, plasminogen, bacteria and platelets.
Human fibrinogen has a strong affinity for fibrin and this association has been exploited to affinity purify fibrinogen. Fibrin immobilised on Sepharose was used to isolate fibrinogen from human plasma (Matthias et al. 1975). Protein structure function studies have identified the peptide sequences in fibrin that have been shown to specifically bind to fibrinogen. Short peptides beginning with the sequence Gly-L-Pro-Arg have been shown to bind fibrinogen (Laudano and Doolittle, 1978). This sequence corresponds to the first three amino acids of the fibrin &agr;-chain exposed by the thrombin catalysed release of the fibrinopeptide A in all vertebrate species. The addition of a second proline to this sequence was later shown to increase the affinity of the peptide Gly-Pro-Arg-Pro for fibrinogen almost ten-fold (Laudano and Doolittle, 1980). Based on this information, synthetic peptides corresponding to these sequences have also been shown to bind to fibrinogen (Gartner and Taylor, 1991).
SUMMARY OF THE INVENTION
The present invention relates to the large scale separation by precipitation of fibrinogen from other blood proteins in human blood plasma, cryoprecipitate, fraction 1 precipitate, other plasma fractions containing fibrinogen or fibrinogen containing culture media produced by recombinant DNA techniques and subsequent treatment of the heparin precipitate. The resultant fibrinogen-enriched preparation may be further purified to homogeneity utilising other precipitation methods, chromatographic steps such as ion-exchange chromatography, affinity chromatography size exclusion chromatography or ultrafiltration.
The present inventors have found that fibrinogen may be recovered from heparin precipitated paste, a by-product from the manufacturing process of Factor VIII (Antihaemophilic Factor, AHF). The heparin precipitate paste may be solubilised with salt containing solutions such as NaCl to provide a fibrinogen preparation of high specific activity. The method of this invention has been shown to be superior to other known isolation procedures in that fibrinogen may be obtained with relative high yield and homogeneity from a discard fraction of processed plasma.
Accordingly, the present invention consists in a method of obtaining a fibrinogen enriched preparation, the method including the following steps:
(i) adding an effective amount of a sulphated polysaccharide (SPS) to a fibrinogen containing solution to form a fibrinogen containing percipitate; and
(ii) extracting fibrinogen from the fibrinogen containing percipitate from step (i) with a solution containing at least 0.1 M, and preferably at least 0.2M, salt to obtain a fibrinogen enriched preparation.
In a preferred embodiment of the present invention the solution includes at least one salt selected from the group consisting of chloride, phosphate and acetate salts, and more preferably includes NaCl. It is preferred that the NaCl is present at concentration of from about 0.1M to about 2.0M, preferably from about 0.2M to about 0.8M.
In a further preferred embodiment the solution includes &egr;-aminocaproic acid.
In another preferred embodiment the SPS is a heparinoid selected from the group consisting of mucopolysaccharide polysulphate, pentosan polysulphate, chondroitin sulphate, dextran sulphate and heparin and is preferably heparin.
The amount of SPS used can be readily determined, however, it is preferred that the SPS is added to the fibrinogen containing solution to provide a concentration of SPS of at least 0.15 mg/ml.
Where the fibrinogen is to be used therapeutically the fibrinogen will be subjected to a viral inactivation step(s). Such inactivation procedures are well known in the art and include heating and solvent detergent treatment.
The fibrinogen containing solution may be any of a number of such solutions well known to those skilled in the art such as plasma (including anti-coagulated plasma), plasma fractions (such as cryoprecipitate and solubilised fraction I) and fibrinogen-containing cell culture media arising from the production of fibrinogen by recombinant DNA techniques. It is, however, preferred that the fibrinogen containing solution is a blood plasma fraction, preferably cryoprecipitate.
The fibrinogen may be further purified from the fibrinogen enriched preparation using any of a range of techniques well known to those skilled in this area. For example, purifying the fibrinogen from the fibrinogen enriched preparation by either reprecipitating the fibrinogen with a protein precipitant in the presence of salts and/or amino acids or by chromatographic techniques such as ion exchange, affinity, hydrophobic or gel permeation chromatography or a combination of both techniques. For use the fibrinogen enriched preparation will typically be treated to remove SPS and/or plasminogen. This can be achieved using a number of methods well known in the art. Examples of known purification methods include those described in the following references, the disclosures of which are incorporated herein by reference:
“Affinity purification of human fibronectin on immobilized gelatine” Regnault V, Rivat C, & Stoltz; Journal of Chromatography, 432 (1988) 93-102
“Isolation of Fibronectin under Mild Conditions” Morgenthaler J, Baillod P & Friedli H; Vox Sang 47 (1984) 41-46
“Plasminogen: Purification from human plasma by affinity chromatography” Deutsch D & Mertz E; Science 170 (1970) 1095-1096
“A Pasteurised Concentrate of Human Plasma Factor XIII for Therapeutic Use” Winkelman L, Sims G, Haddon M, Evans D & Smith J; Thrombosis and Haemostasis 55(3)(1986) 402-405
“The Preparation of Human Fibrinogen Free of Plasminogen” Mosesson M; Biochim Biophys Acta 57 (1962) 204
Demaria Grace
Goss Neil
Kanellos Jerry
Martinelli Teresa
Carlson Karen Cochrane
CSL Limited
Nixon & Vanderhye
Snedden Sheridan
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