Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Utility Patent
1998-01-23
2001-01-02
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Utility Patent
active
06168938
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the preparation of thrombin by the activation of prothrombin. Thrombin prepared in accordance with this invention may be used in a fibrin glue kit, as a topical agent in its own right for therapeutic use in humans, or as a diagnostic reagent.
BACKGROUND OF THE INVENTION
Thrombin is a serine protease representing the active form of the coagulation factor, prothrombin. Its most studied role is its ability to convert soluble fibrinogen into an insoluble fibrin clot, thereby stemming the flow of blood from an injured blood vessel. Thrombin also cleaves and activates factor XIII to factor XIIIa which serves to stabilise the formed fibrin clot even further (Furie, 1991).
The conversion of prothrombin to thrombin can be performed in many different ways. According to the classic description of coagulation, prothrombin is converted to thrombin by coagulation factor Xa in the presence of cofactors factor Va, phospholipid and calcium chloride (Esmon, 1974). Various snake venoms also cleave prothrombin to form thrombin, examples being the venom of the Taipan snake
Oxyuranus scutellus
(Owen and Jackson, 1973), the Tiger snake
Notechis scutatus
(Jobin and Esnouf, 1966), and the saw-scaled viper
Echis carinatus
(Franza, 1975). However, none of these have been used commercially in the production of thrombin for clinical application.
Factor Xa, required for classic prothrombin activation, can be generated in many ways. The Intrinsic Pathway of Coagulation suggests that factor X is converted to factor Xa when incubated with factor IXa in the presence of cofactors factor VIIIa, phospholipid and calcium chloride. The Extrinsic Pathway of Coagulation is initiated by the exposure of tissue factor (containing thromboplastin) to factor VII in the presence of calcium chloride. This serves to activate factor VII to factor VIIa which in turn converts factor X to factor Xa. Both forms of factor Xa then activate prothrombin as described above (Furie, 1991) There are no reports of commercial preparations of thrombin exploiting features of the Intrinsic Pathway for generation of factor Xa, however there are a number of reports of exploitation of the Extrinsic Pathway to generate sufficient factor Xa to subsequently activate prothrombin by adding thromboplastin to the prothrombin-containing starting material (see, for example, Japanese Patent Publications Nos 63290829 and 2019400 in the name of Green Cross Corporation, and European Patent Publication No. 443724 in the name of Baxter International Inc.) Factor Xa can also be formed by incubating factor X with Russell's Viper venom (RVV) in the presence of calcium chloride (Kisiel, 1976). This approach has been used commercially (Bio-Products Laboratories) for the preparation of thrombin as a diagnostic reagent. However, the need to demonstrate the absence of soluble RVV in the final product would pose difficulties with its registration as a therapeutic agent.
A crude preparation of thrombin has been manufactured by the present applicant using thrombogenic discard fractions from the Prothrombin Complex Concentrate (PCC) manufacturing process, in which the prothrombin has been converted to thrombin by incubating the starting material with human placenta-derived thromboplastin in the presence of calcium chloride. A limitation of this method has been controlling the quality control of the placental thromboplastin. As a diagnostic product, this has not posed such a problem, however, for clinical use, clinical registration requires strict safeguards which have not been practical to introduce.
Commercial preparations of thrombin are of particular value as therapeutic agents as well as in diagnostic applications. In particular, thrombin may be used to promote coagulation as a topical agent (in either liquid or powderform) or incorporated into wound dressings. Furthermore, thrombin may be used as a component of a fibrin glue kit in which, in use, thrombin and fibrinogen factor XIII are combined for application to surgical incisions and other fresh wounds to replace the use of sutures, particularly during surgery on delicate tissues.
In work leading to the present invention, the present inventors have developed a method of activation of prothrombin to thrombin which does not require the use of thromboplastin, either human or any other species. Furthermore, the method of the present invention is characterised by the use of a fraction from the Prothrombin Complete Concentrate (PCC) prepared by the published method of Middleton et al, 1973 which has normally been discarded. As a result, the present invention provides a thrombin product which can be produced economically on a commercial scale.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method for the preparation of thrombin from a prothrombin-containing material, characterised in that the prothrombin-containing material is an Activated Prothrom bin Complex Concentrate (ActPCC) fraction as described hereinafter, and said ActPCC fraction is contacted with divalent cations in order to convert prothrombin in said fraction to thrombin. As mentioned previously, it has been found that when thrombin is prepared from the ActPCC fraction in accordance with this invention, the presence of thromboplastin is not required.
The “ActPCC fraction” which is used as the prothrombin-containing starting material in accordance with the invention comprises at least some of the thrombogenic fractions in the ascending or pre-peak material produced during the preparation of Prothrombin Complex Concentrate (PCC) by elution of the bound proteins from Cohn fraction I supematant from DEAE-cellulose resin. Further details of the ActPCC fraction, and the production thereof, are given in the detailed description and the Examples hereunder.
The present invention also extends to thrombin prepared by the method broadly described above, as well as to therapeutic and diagnostic preparations thereof. In addition, the invention extends to fibrin glue kits comprising thrombin produced by the method of this invention.
Preferably, the divalent cations used in the method of this invention are Ca
2+
ions, and conveniently CaCl
2
is used in the activation of the ActPCC fraction.
In another preferred aspect, it has been found that the optimum concentration of Ca
2+
ions for production of thrombin in accordance with this invention is in the range of 25 to 50 mM, more preferably from 35 to 50 mM, and most preferably at a concentration of about 40 mM. Furthermore, it has also been found that although the production of thrombin can be performed at temperatures in the range of 4° C. to 37° C., the optimum temperature does vary with Ca
2+
concentration. In particular, the optimum and hence preferred temperature is in the range of 22° C. to 25° C.
In a particularly preferred embodiment of this invention, the ActPCC fraction is treated with 40±5 mM CaCl
2
at 22-25° C. for a period of 4-5 hours.
Of course, after the treatment in accordance with this invention to convert the prothrombin to thrombin, the thrombin is recovered and purified by methods which are well-known in the art in order to produce a commercial concentrate which may, for example, be >90% pure thrombin.
Preferably, the further processing steps include a virus-inactivation step, such as a solvent/detergent treatment (for example by incubating the product with 0.3% TNBP/1% Tween 80 for 8-12 hours at 24° C.-26° C.), in order to produce a virus-safe thrombin concentrate. The virus-inactivated product may then be further treated for recovery of the thrombin by chromatography, for example with Heparin-agarose (such as Heparin-Sepharose) at a pH in the range of 6-8 (preferably pH 7.5), with elution of thrombin by NaCl, preferably at a NaCl concentration above 350 mM.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but
Kanellos Jerry
Kupczyk Malgorzata
Oates Adrian Malcolm
CSL Limited
Foley & Lardner
Lilling Herbert J.
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