In the living organism, a plurality of proteins and enzymes is produced by the cellulary biosynthesis as inactive pre-stages (pro-proteins (precursor proteins) or pro-enzymes). If required, these inactive pre-stages are then converted into their active forms, e.g. by limited proteolysis. Thus, in the human body, prothrombin is converted to thrombin by the protease factor Xa in a prothrombinase-complex reaction. Inactive factor X is converted to active factor Xa, by the protease factor IXa, e.g.
The recovery of the activated proteins is of major interest both for clinical applications as well as for diagnostic purposes. The activated proteins in their pure form may then, e.g., be used to control other proteolytic processes, such as the blood coagulation by thrombin in surgery, for the therapy or diagnosis or for the recovery of specific antibodies.
From living organisms, such as, e.g., human blood, the active proteins can be recovered in very limited amounts only. Therefore, the pro-proteins or pro-enzymes are mostly converted to the activated forms by the action of suitable proteases in vitro. Such a method is known from EP-0 378 798. According to this method, prothrombin derived from human plasma is adsorbed on a solid carrier, and the material is treated with Ca.sup.2+ ions, and thereby, together with proteases present in the plasma, the conversion of prothrombin to thrombin is effected.
Another method is described in EP-A-0 565 511, in which, according to a preferred embodiment, the pro-protein to be activated is immobilized on a carrier and then is converted to the active enzyme by a soluble protease. To render this conversion controllable, it is carried out in the presence of a detergent or of a chaotropic substance. For the recovery of the activated proteins, however, further purification steps are required, in particular for separating the protease again.
According to EP-A-0 541 507, prothrombin in soluble form is converted to thrombin by adding coagulatively active salts to a prothrombin-containing solution. Subsequently, thrombin is further purified by ion exchange chromatography and/or affinity chromatography.
According to EP-A-0 565 512, thrombin is prepared by treating a prothrombin-containing solution with immobilized trypsin for 150 minutes. There, the use of the protease in the immobilized form allows for an easier separation of the protease after activation.
According to EP-A-0 416 890, recombinant human protein C (rHPC) is activated by a 2 hour treatment with thrombin immobilized on glass beads. Separation of activated rHPC from immobilized thrombin is effected by centrifugation.
When using proteases for the inactivation of pro-enzymes or pro-proteins, the problem has been encountered that the process of proteolysis does not end at the stage of the active enzymes. Rather, further peptide bonds are continued to be hydrolysed by the protease in the course of the reaction, and the activated proteins are further cleaved to low-molecular peptides that are inactive again (Kisiel and Hanahan, 1973, Biochim. Biophys. Acta 329, 221-232). Therefore, in such methods, the yield is known to be low, when activating prothrombin to thrombin by trypsin, e.g., the yield is only 50% (Kisiel and Hanahan, 1973, Biochim. Biophys. Acta 329, 221-232).
Attempts have been made to improve such methods by adding stabilizers, such as albumin or glycine, to the activating reaction, so as to reduce the further degradation of the activated proteins (Landaburu et al. 1961, Am. J. Physiol. 201, 298-302).
However, a substantial disadvantage of these methods consists in that, although at a slower pace, a further degradation to protein fragments does occur, and protein mixtures form which additionally contain large amounts of additives, such as detergents or glycerol. Therefore, complex methods of purifying the activated enzymes are necessary.
Protein fragments and detergents or chaotropic substances were removed according to EP-A-0 565 511 by relatively complex chromatographic methods.
USPTO Backgrounds
USPTO Backgrounds