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
1997-05-30
1998-11-03
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...
530413, 530416, 530417, 530427, 530830, 530831, 424 9467, 424529, 424532, 435183, 435184, 514 2, 514 6, 514 21, A61K 3514, A61K 3846, A23J 100
Patent
active
058310266
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
Proteases generally tend to reduce the activity of coagulation factor VIII by degrading the molecule. The present invention relates to a process for reducing the detrimental influence of metal-dependent proteases upon recombinant factor VIII molecules, by adding an inhibitor of metal-dependent proteases to a solution after harvest. The solution is suitably the harvest solution or any aqueous solution being fed to or leaving the primary isolation, being the initial step of the purifying sequence. The inhibitor is selected from i) complexing agents with a stronger affinity for the alkaline-earth metal ion or metal ion of the protease than for the ion or ions stabilizing the factor VIII molecule and ii) compounds structurally related to the natural substrate of the protease and containing an electronegative moiety. The presence of an inhibitor, preferably a complexing agent, after harvest allows for a prolonged harvest period and considerably higher yield with essentially retained factor VIII activity. The invention further relates to an aqueous solution containing recombinant factor VIII which has been purified according to the present process and use of such an aqueous solution, for the manufacture of a medicament for administration to a patient having the symptoms of hemophilia. Also, the invention relates to a method for treatment of hemophilia by administration of a therapeutically effective amount of recombinant factor VIII which has been purified according to the present process.
BACKGROUND OF THE INVENTION
Hemophilia is an inherited disease which has been known for centuries, but it is only within the last four decades that it has been possible to differentiate between the various forms; hemophilia A and hemophilia B. Hemophilia A is the most frequent form. It affects only males with an incidence of one or two individuals per 10 000 live-born males. The disease is caused by strongly decreased level or absence of biologically active coagulation factor VIII (antihemophilic factor), which is a protein normally present in plasma. The clinical manifestation of hemophilia A is a strong bleeding tendency and before treatment with factor VIII concentrates was introduced, the mean age of the patients concerned was less than 20 years. Concentrates of factor VIII obtained from plasma have been available for about three decades. This has improved the situation for treatment of hemophilia patients considerably and offered them the possibility of living a normal life.
Until recently, therapeutic factor VIII concentrates have been prepared by fractionation of plasma. However, there are since some years methods available for production of factor VIII in cell culture using recombinant DNA techniques as reported in e.g. W. Wood et al, Nature, 312, p. 330-37 (1984) and EP-A-0 160 457.
It is well known that proteins are cleaved by proteases, such as serine, cysteine, aspartic and metallo proteases. Many proteases need alkaline-earth metals or metals (in the following just denoted metals) for their activity. The metal-dependent proteases are either considered to be metal-activated proteases (to which metal ions must be added for activity) or metallo proteases (which contain metals as an integral part of their structure). Concerning the first group, activation and stabilization of enzymes by metals frequently occur in several classes of proteases, such as serine and cysteine proteases.
The importance of a metallo protease in cultured endothelial cells for the secretion of a certain metabolite has been shown by R. Ikegawa et al in Biochem. Biophys. Res. Comm. 171(2), p. 669-675 (1990). This was revealed by the suppressing effect on this secretion recognized by the addition of a metallo protease specific inhibitor. It was evident, however, that the enzyme was confined to the intracellular space, since no effect of the inhibitor was obtained in a cell-free conditioned medium.
The effect of proteases are though far more often mentioned in the context of the potential risk of degradation of the protei
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Almstedt Annelie
Ostlin Anna
Sandberg Helena
Smeds Anna-Lisa
Wrangel Maria
Mohamed Abdel A.
Pharmacia & UpJohn AB
Tsang Cecilia J.
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