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
1990-04-06
1993-10-12
Russel, Jeffrey E.
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, 530383, 530395, 530416, C07K 322, C07K 1506
Patent
active
052527093
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the separation of proteins of a fraction of human or animal plasma by anion-exchange chromatography using a technique that makes it possible to obtain, in a single stage, a very high purification rate, in particular for Factor VIII, for fibrinogen and for Von Willebrand's factor.
The provision of blood proteins for therapeutic purposes necessitates the use of purification techniques that make it possible to obtain products of high purity and completely free of contaminants, particularly other proteins or substances of foreign origin such as antibodies.
It is essential, for instance, in treating haemophilia A, to have at one's disposal Factor VIII concentrates of very high purity; indeed, the patients are given numerous, repeated injections of Factor VIII concentrates and, at the same time, substantial quantities of fibrinogen and immunoglobulins which can induce undesirable immune responses. Hence, repeated injections of insufficiently purified Factor VIII can be carried out only with plasma concentrates of the same group in order to avoid typical transfusion accidents due to differences in blood group and caused by the presence of immunoglobulins.
Factor VIII concentrates are most often prepared from a fraction of cryoprecipitated human plasma. The purity of Factor VIII concentrates, generally obtained from industrial scale human plasma processing centers, is often in the order of 1 IU/mg and does not generally exceed the limits of 10 to 20 IU/mg. Conventional production techniques make use of precipitation stages that aim to eliminate, often very inadequately, protein contaminants such as fibrinogen, fibronectin and immunoglobulins. These techniques can use or combine precipitation at low temperature (10.degree. C.), or the addition of protein precipitating agents; hydrophilic polymers such as PEG (Newman et al., Br. J. Haematol 21:1-20, 1971; Hao et al., in Methods of Plasma Protein Fractionation, Academic Press 1980, PP. 57-74), polyvinylpyrrolidone (Casillas and Simonetti, Br. J. Haemato, 50:665-672, 1982), dextran, Ficoll, Percoll, hydroxyethylated starch and albumin have thus been proposed as Factor VIII precipitating agents (Farrugia et al., Thromb Haemostas, 51:338-342, 1984). The same applies to the use of glycine and sodium chloride recommended by Thorell and Blomback. Similarly, some authors (Ng et al., Thrombosis Res., 42:825-834, 1986) have succeeded in combining three precipitating agents, namely: PEG, glycine and sodium chloride, to obtain Factor VIII concentrates having a specific activity of between 10 and 16 IU/mg.
Use has also been made of steric exclusion chromatography, or gel filtration, techniques aimed at recovering a fraction of high molecular weight containing the Factor VIII:C--Von Willebrand's factor complex partially free of fibrinogen. This technique provides, at a low rate of output, a product the specific activity of which does not exceed 30 IU/mg and which necessitates the addition of albumin as a stabilizer (leading to a drop in specific activity to approximately 3 to 5 IU/mg). This technique poses a number of problems regarding adaptation to large scale production as it is difficult to maintain the resolving power of industrial gel filtration columns over a period of time.
Factor VIII concentrates have also been produced by incorporating in the production program contact with microballs of porous silicon dioxide designed to imprison protein contaminants of low molecular weight (Margolis et al., Vox Sang. 46:341-348, 1984). The specific activity of the product remains relatively small: 1 IU/mg.
New techniques have recently made their appearance in the preparation of very high purity Factor VIII concentrates. The firms of Hyland and Travenol, for instance, have proposed concentrates obtained using immune affinity chromatography methods (Zimmerman and Fulcher, Thrombosis Res., Suppl. VII, p. 58, 1987; Berntorp and Nilsson, Thrombosis Res., Suppl. VII, p.60, 1987; Levine et al., Thrombosis Res, Suppl. VII, 1987). These techniques consist in p
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Burnouf Thierry
Burnouf-Radosevich Miryana
Centre Regional de Transfusion Sanguine de Lille
Russel Jeffrey E.
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