Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-08-04
2004-01-13
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
C530S380000, C530S381000, C530S415000, C435S007700, C424S094100, C424S094600
Reexamination Certificate
active
06677440
ABSTRACT:
This application claims priority from German patent application No. 19937219.5 filed Aug. 6, 1999.
The invention relates to a process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins, and of pharmaceutical preparations which contain the proteins mentioned individually or as a mixture.
German patent application 19 903 693.4 has already disclosed a protease for the activation of blood clotting factor VII, a process for its production, for its detection and for its inactivation, and pharmaceutical preparations which contain this protease. This protease, first isolated from plasma, occurs there together with a nonactivated form, which is designated below as “proenzyme”. The protease activates blood clotting factor VII and accelerates clotting, as has been shown by numerous experiments. In the further investigation of the biological properties of this protein, identified as serine protease, it emerged that singlechain plasminogen activators, such as prourokinase, are also effectively activated. Moreover, inactivation of factors V and VIII in vitro was observed. In addition to the sequenced regions already described in German patent application 19 903 693.4, N-terminal sequencings of protease fractions were carried out. The following amino acid sequences characterize the FVII-activating prpotease: TYGGFKSTAGKHP (SEQ ID NO:1); LLESLDPDXTPD (SEQ ID NO:2); EFHEQSFRVEKI (SEQ ID NO:3); SKFTXAXPXQFK (SEQ ID NO:4); where X means not identified. The sequences of the protease mentioned elucidated up to now show that they agree 100% with sequences of the protease published by Choi-Miura (Choi-Miura et al. J. Biochem. 1996; 119: 1157 to 1165).
The investigations until now have especially concentrated on the protease in its activated form. The inactive form of the protease present in the plasma as a proenzyme was only recently discovered by means of a protein band pattern in the SDS-PAGE after reduction of the sample. Since, on the activation of the protease, cleavage and thus activation take place at a site of the primary structure typical for serine proteases two or more bands are visible on electrophoresis. On reduction of the chains which are connected by disulfide bridges, the individual bands become recognizable in accordance with their lower molecular weight, the proenzyme remaining as a large individual chain. This also became clear in more complex solutions after transfer of the proteins to membranes and subsequent Western blotting using suitable antibodies.
For therapeutic reasons, there is now an interest in having available both the protease in its activated form and the proenzyme, in addition to the mixture of the two proteins mentioned. Whereas the activated protease can be used for the rapid activation of blood clotting factor VII or the single-chain plasminogen activators in order to influence acute syndromes, the proenzyme form of the protease is especially to be chosen as a preferred agent for medium- to longer-term prophylaxis or treatment of inherited or acquired deficiency states or alternatively for increasing the plasma level beyond the physiological extent. However, it is to be taken into account here that the stabilization of an activated protease is difficult, since, for example, self-degradation can take place or the molecule can be unstable on account of its structural conditions. Previous studies have shown that the protease activating factor VII can be isolated and stabilized in its proenzyme form only under special circumstances.
The previous investigations have shown that the biological activities of this protease can be increased by calcium and/or heparin or substances related to the latter. This property has already been previously used in order to adsorb the protease on immobilized heparin and to obtain an enriched fraction. Moreover, it is already known that anion-exchange chromatography is also suitable for the purification of the protease. The combination of both purification steps is suitable for obtaining the protease in enriched form. An aprotinin matrix can also be used for the preparation in pure form of the activated protease.
A process for the preparation in pure form and simultaneous stabilization of the protease activating blood clotting factor VII and/or its proenzyme has now been found, in which they are obtained from biological fluids or those obtained in the case of preparation by genetic engineering by
a) anion- and/or cation-exchange chromatography at a pH value below the isoelectric point of the protein to be isolated or
b) a combination of anion- or cation-exchange chromatography with affinity chromatography
and/or fractional precipitation at pH values between 2.5 and 9.0, preferably between 2.5 and 7.2, the affinity chromatography being carried out using calcium phosphate/hydroxyapatite
a hydrophobic matrix,
a chelate matrix,
a matrix that is coated with an immobilized monoclonal or polyclonal antibody directed against the protein to be isolated, or its F(ab) or F(ab)
2
fragments.
A particularly suitable method for the preparation in pure form of the protease and/or its proenzyme is anion- and/or cation-exchange chromatography. The use of these methods for the preparation in pure form of the activated protease was admittedly proposed earlier, but the process conditions previously used did not produce completely satisfactory results. This is principally due to the fact that the risk of the activation of the proenzyme on contact with the surfaces of the matrices is very high. The object was therefore to develop a process which makes possible the preparation of the activated protease and of the proenzyme in a pure and stable form.
Surprisingly, it was then shown that very low pH values, in particular pH values between 2.5 and 7.2, damage neither the activated form of the protease nor the proenzyme and can therefore be employed to good effect in the adsorption and elution. By this means, almost trouble-free handling of the protease activating factor VII is made possible, since most other proteases circulating in the plasma are not active or only very slightly active in the acidic medium and the risk of proteolytic activation is thus minimized. The danger of the self-degradation of the protease is also decreased in this way. Since, as is known, extremely acidic pH values could include the risk of denaturation and thus bring about a loss in activity of the protease, the activity of the protease obtained from a strongly acidic medium occurring in the case of action on chromogenic substrates. It was shown in this case that both the protease and its proenzyme can be handled without loss of activity in the short term, up to a pH of 2.0. At a pH of 2.5 to approximately 7.2, the protease and its proenzyme can be stored for several months, the highest stabilities being observed at a pH of below 6.5.
In this case it was surprisingly found that anion- and/or cation-exchange chromatography at the low pH values mentioned can be used for the purification of the protease and of the proenzyme. This is so remarkable because in this case adsorption is possible on the exchanger matrices at pH values which lie below the isoelectric point of the protease or the proenzyme.
Up to now, there is still no scientific explanation of the interactions on which this adsorption is based. However, it is possible by means of this adsorption in the acidic medium to remove a large number of impurities that do not bind to the matrices at these pH values. A considerable enrichment of the protease on the matrix is thus achieved. After washing the matrix, the protease and/or the proenzyme can be eluted by an increase in the ionic strength.
For the preparation in pure form of the above mentioned proteins, the anion- and/or cation-exchange chromatography can be combined with chromatography and/or fractional precipitation. Independently of whether the above mentioned purification processes are employed individually or in any desired combination, it is recommended additionally to add protease inhibitors
Feussner Annette
Roemisch Juergen
Stoehr Hans-Arnold
Aventis Behring GmbH
Carlson Karen Cochrane
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Lin Samuel W.
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