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
2003-12-30
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
C530S381000, C530S300000, C530S350000, C514S002600, C435S069200, C435S069600
Reexamination Certificate
active
06670455
ABSTRACT:
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 was shown by numerous experiments. In the further investigation of the biological properties of this protein, identified as serine protease, it emerged that single-chain 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 protease: IYGGFKSTAGKHP; LLESLDPDXTPD; EFHEQSFRVEKI; SKFTXAXPXQFK; 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, a cleavage at a site of the primary structure typical for serine proteases and thus activation takes place, two or more bands are visible on electrophoresis. On reduction of the chains which are connected by disulfide bridges, the individual bands become visible in accordance with their lower molecular weight, the proenzyme remaining as a large individual chain. This was also 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 showed that the protease activating factor VII can only be isolated and stabilized in its proenzyme form under special circumstances.
The investigations until now have shown that the biological activities of this protease can be increased by calcium and/or heparin or substances related to this. 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 of the protease activating blood clotting factor VII and/or its proenzyme has now been found, in which one or more affinity chromatography separation processes and/or fractional precipitation are employed.
Affinity chromatography separation processes which can be used are adsorption on
calcium phosphate/hydroxyapatite,
a hydrophobic matrix,
a chelate matrix,
a matrix on which heparin or a substance related to heparin, such as heparan sulfate or dextran sulfate, is immobilized, and/or a matrix which 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 simple and rapid method for the enrichment of the protease and of the proenzyme is in this case adsorption on calcium phosphate/hydroxyapatite. In the course of this, the solution which contains the protease and the proenzyme is mixed with calcium phosphate at a pH of between 2.5 and 9.0, preferably between 2.5 and approximately 7.2. After subsequent sedimentation, e.g. by centrifugation or by filtration, the sediment, optionally after resuspending one or more times in a buffer solution, is eluted with addition of, for example, 0.2 M sodium citrate. The protease and the proenzyme are then found in the eluate.
The adsorption of the protease on hydrophobic matrices or on hydrophobic ligands which are coupled to appropriate matrices can also be used according to the invention. Examples are phenyl- and octyl-sepharoses or a phenylalanine coupled to a matrix. The bound protein is eluted in a manner known per se using a buffered solution of low ionic strength, which can contain phenylalanine, glycerol or ethylene glycol.
Since the protease and the proenzyme enter into an interaction with cations, especially with calcium and magnesium ions, which is confirmed by an increase in their activity in the presence thereof, chromatography by means of so-called “chelate matrices” suggests itself for enrichment thereof from corresponding solutions. Chelate compounds with zinc, copper or nickel ions are particularly suitable here. After the washing of the matrix loaded with the protease, an imidazole buffer, if appropriate with a linear gradient, can also be employed for the elution of bound proteins.
The process according to the invention can also be carried out by removing the said proteins by fractional precipitation, e.g. by addition of polyethylene glycol or ammonium sulfate, from the liquids containing them. Fractional precipitations of this type can be employed as the sole separation process, but the yield and the effectiveness of the process according to the invention are further improved if it is combined in any desired sequence with other purification processes known per se. It is thus possible, by admixture of polyethylene glycol (preferably PEG 6000) from 10% final concentration to a solution containing the protease activating factor VII and the proenzyme in the pH range from 2.5 to 9.0, to carry out a precipitation of the protease and of the proenzyme without a loss of activity occurring in the course of this. Owing to fractional PEG precipitation which is achievable thereby, separation of impurities is possible. This also applies to fractional precipitation by means of ammonium sulfate, from approximately 15% final concentration. The protease precipitates obtained can not only be stored without loss of activity, they are particularly suitable for the concentration of the protease and of the proenzyme, so that the preparation in pure form of the proteins mentioned is possible in a shorter time and, moreover, exposure of the protease to activating surfaces which lead to losses of activity, such as occurs, for example, in the use of filters, is avoided.
In general, however, it is expedient to carry out all process steps for the isolation of the protease and of the corresponding proenzyme from a solution containing these proteins, such as plasma, plasma fractions, tissue fluids or cell culture supernatants of the recombinantly or transgenically
Feussner Annette
Roemisch Juergen
Stoehr Hans-Arnold
Aventis Behring GmbH
Carlson Karen Cochrane
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Liu Samuel W.
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