AU781717B2 - Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or mixture of both proteins by means of ion-exchange chromatography - Google Patents

Description

IML

r'/uu/lv I IL51i Regulation 3.2(2)

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: PROCESS FOR THE PREPARATION IN PURE FORM OF THE PROTEASE ACTIVATING BLOOD CLOTTING FACTOR VII, ITS PROENZYME OR A MIXTURE OF BOTH PROTEINS BY MEANS OF ION- EXCHANGE CHROMATOGRAPHY The following statement is a full description of this invention, including the best method of performing it known to us 1- AVENTS BEHRING GMBH 1999/Z011 Ma 1212 C41 Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins by means of ion-exchange chromatography 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 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).

2 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 longerterm 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 Sprotease 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 19/04 '05 TUE 12:05 FAX 61 2 9888 7600 WATERMARK 009 3 purification of the protease. The combination of both purification steps is suitable for obtaining the protease in enriched form. The use of 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 below the isoelectric point of the protein to be isolated or b) a combination of anion- or cation-exchange chromatography with 15 affinity chromatography 0 and/or fractional precipitation at pHs of between 2.5 and 9.0, preferably between 2.5 and 7.2, the affinity chromatography being carried out using calcium phosphate/hydroxyapatite 00 20 a hydrophobic matrix, a chelate matrix, 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.

00 Therefore disclosed in herein is a process for the preparation in pure form of at least one of the protease activating blood clotting factor VII or its proenzyme from biological fluids or genetically engineered preparations: a) by at least one of anion- or cation-exchange chromatography at a pH below the isoelectric point of the protein to be isolated, or b) by at least one of: COMS ID No: SBMI-01211178 Received by IP Australia: Time 12:13 Date 2005-04-19 21/04 '05 THU 11:43 FAX 61 2 9888 7600 WATERMARK (0011 3a i) a combination of anion- or cation-exchange chromatography with affinity chromatography, or ii) fractional precipitation at pHs of between 2.5 and the affinity-chromatography separation processes being carried out either using: calcium phosphate/hydroxyapatite, and/or a hydrophobic matrix, and/or a chelate matrix, 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; wherein the protease activating blood clotting factor VII: a) is inhibited by the presence of aprotinin, b) is increased in its activity by calcium ions and/or heparin or 15 heparin-related substances and Sc) in SDS-PAGE, on subsequent staining in the non-reduced 0 state, has one or more bands in the molecular weight range from to 75 kDa and in the reduced state has a band at 40 to 55 kDa and one or more bands in the molecular weight range from 10 to kDa.

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 25 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 COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21 4 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 pHs, in particular pHs between 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 pHs 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 •was measured, for example, by a photometric determination of the extinction 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 In this case it was surprisingly found that anion- and/or cation-exchange chromatography at the low pHs 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 pHs which lie below the isoelectric point of the protease or of the proenzyme.

Up to now, there is still no scientific explanation of on which interactions this adsorption is based. However, it is possible by means of this adsorption in the acidic medium to remove a large number of impurities which do not bind to the matrices at these pHs. 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.

5 For the preparation in pure form of the abovementioned proteins, the anionand/or cation-exchange chromatography can be combined with affinity chromatography and/or fractional precipitation. Independently of whether the abovementioned purification processes are employed individually or in any desired combination, it is recommended additionally to add protease inhibitors in all process steps, in order to block cleavage of the proenzyme into the activated protease. Suitable protein stabilizers which are added in this case are solubilizers, preferably hydroxyproline, detergents, preferably Tween® or Triton®, proteins, preferably albumin, gelatin, fibronectin and vitronectin or similar proteins, reductants, preferably dithiothreitol, mercaptoethanol or cystiene, and/or proteinase inhibitors such as aprotinin, c-2-antiplasmin, C1-esterase inhibitor, inter-a-trypsin inhibitor, antithrombin IIl/heparin or synthetic inhibitors.

A particularly good stabilization can be achieved during the preparation in pure form of the protease and of the proenzyme if, as further protein stabilizers, -complexing agents of divalent ions, preferably EGTA, EDTA or citrate and/or divalent ions, preferably calcium ions, and/or amino acids, preferably glutamate, arginine, lysine or glycine, and/or sugars, preferably glucose, arabinose, mannose or mannitol, and/or 6 alcohols, preferably ethylene glycol or polyethylene glycol, are added.

The abovementioned process steps can also additionally be combined with fractional precipitation of the protease and/or its proenzyme from its solution, which is carried out by addition of polyethylene glycol from a concentration of at least 10% by weight or ammonium sulfate from a concentration of at least 15% by weight.

It is particularly worthy of note that in the processes described above the proenzyme form of the protease can also be obtained in pure form. As a matter of fact, it was seen that ion-exchange chromatography, under the said acidic conditions, using a solution which especially contained the proenzyme, led to an eluate which contained the proenzyme exclusively or at least to a very greatly enriched extent. In this case, the nativity of the proenzyme thus obtained can be **determined with the aid of one of the activity tests which are described in German patent application 196 26 531.3. for example, by the photometric determination of the extinction occurring on action on chromogenic substrates or by the single-chain formation occurring after reduction of the sample, which can be detected by SDS-PAGE/Western blotting. This shows that according to the invention the preparation of the proenzyme is possible in a rapid and efficient manner and with a high yield.

When using the abovementioned process steps, it is thus possible to obtain both the purified protease activating factor VII, its proenzyme or alternatively a mixture of the activated protease and the proenzyme. A route which is particularly worthy of recommendation for the preparation of a pure activated protease consists in the chromatographic separation of the protease activating factor VII from its proenzyme by means of stepwise elution, in which a substance -7 is immobilized on the support material which has bonds of different strength to the protease on the one hand and to the proenzyme on the other hand. Different eluates can thus be obtained which contain either only the activated protease or only the proenzyme.

To obtain the proenzyme, an inhibitor with strong affinity for the activated protease is in this case immobilized on the matrix. Serine protease inhibitors, in particular C1 esterase inhibitor, A-2 antiplasmin, antithrombin III (with admixture of heparin to the application solution) or low molecular weight, high-affinity inhibitors, which can also be of synthetic nature, are especially suitable for this.

The proenzyme which is not bound or less firmly bound to the matrix is then found in the solution flowing through the column. On the other hand, for the .*preparation in pure form of the activated protease, an inhibitor is immobilized on •the matrix which only has a weak inhibitory potential and reversibly binds the activated protease. In this case, after washing off the proenzyme, the active protease bound to the matrix is eluted and can then be obtained in pure form. A suitable support material for this process variant is, for example, one treated with aprotinin or low molecular weight, reversible inhibitors, which can also be of *.synthetic nature.

Of course, antibodies or fragments thereof, which can differ between the activated protease and the proenzyme, can also be used for the preparation of the said proteins in pure form. For this, monoclonal antibodies can especially be employed which can recognize "neoepitopes" after activation of the protease, for example, the activation or cleavage site of the protein.

Under the affinity-chromatography processes to be employed according to the invention, adsorption on calcium phosphate/hydroxyapatite is to be emphasized as a simple and rapid method for the enrichment of the protease and/or of the proenzyme. In this case, the solution which contains the protease and the proenzyme is mixed with calcium phosphate at a pH of between 2.5 and preferably between 2.5 and 7.2. After subsequent sedimentation, e.g. by 8 centrifugation or by filtration, the sediment, if appropriate 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- or octyl-Sepharoses® or a phenylalanine coupled to a matrix. The elution of the bound protein is carried out in a manner known per se using a buffered solution of low ionic strength, which can contain phenylalanine, glycerol or ethylene glycol.

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Since the protease and the proenzyme enter into an interaction with cations, especially with calcium and magnesium ions, which is confirmed by an increase ooo.oi 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 in this case. After the washing of the matrix loaded with the protease, an imidazole buffer can also be employed for the elution of bound proteins, if appropriate with a linear gradient.

The process according to the invention can also contain an affinitychromatography purification step in which the support material employed is a matrix on which monoclonal or polyclonal antibodies directed against the said proteins, or their F(ab) or their F(ab) 2 fragments or other substances suitable for the reversible binding of the said proteins, are immobilized.

The protease obtained by the process according to the invention, its proenzyme or the mixture of both proteins can be stored without loss of activity in the acidic pH range at pHs of approximately 2.5 to 7.2 with or without the addition of additional stabilizers, while in the alkaline range addition of stabilizers is necessary.

9 Therapeutically, the said activated protease, the proenzyme or the mixture of both compounds can be used to assist blood clotting in the case of a tendency to bleeding, in the case of absence of factors of the endogenous clotting branch or as FEIBA factor VIII bypassing activity), but also for the endogenous and exogenous activation of plasminogen activators such as prourokinase or singlechain tPA. This activity can also be employed in combination with single-chain or double-chain plasminogen activators or anticoagulants by use of the said protease for the prophylaxis or therapy of thromboembolic disorders. Syndromes which are associated with thrombotic complications, such as cardiac infarct, angina pectoris, stroke or leg vein thromboses, can thus be successfully treated.

A further subject of the invention is therefore a pharmaceutical preparation which contains an amount of the protease activating blood clotting factor VII and/or its proenzyme form sufficient for the dissolution of fibrin-containing thrombi. This preparation can also moreover contain single-chain plasminogen activators and/or anticoagulants. Expediently, a proteinase stabilizer or a reductant such as dithiothreitol, mercaptoethanol or cystienee is additionally added to the preparation in order to reduce the risk of polymer formation during processing or on storage.

Fibrinolytic processes also play a part in wound-healing processes. In this case, the said protease and/or the proenzyme can be administered intravenously or locally, subcutaneously, intradermally, intramuscularly or, in the case of injuries and wounds, as a constituent of a fibrin adhesive or alternatively topically or bound to a suitable carrier matrix, e.g. in the form of a web or of a patch, where combination with growth factors can be expedient. In general, a pharmaceutical preparation of this type is used in liquid or lyophilized form, to which protein stabilizers known per se can be added, for example, complexing agents, divalent cations such as calcium, amino acids such as glutamate, arginine, lysine or glycine and/or sugars such as glucose, arabinose, mannose or mannitol.

10 Moreover, the protease and/or its proenzyme can also be employed for the coating of articles, consisting of plastics or metals, to be implanted in the body, such as synthetic heart valves, blood vessels, but also cannulas inserted for taking blood or for artificial feeding.

The invention is illustrated by the following examples: S* 11 Example 1 Preparation in pure form by means of immobilized monoclonal antibodies Monoclonal antibodies which are directed against the protease activating factor VII were coupled to BrCN-Sepharose®. 30 ml of this mAb matrix were packed into a column and the resin was equilibrated with 50 mM sodium citrate, 0.1 M sodium chloride (NaCI), 0.1 M arginine x HCI, pH 100 ml of citrate plasma were pumped through the column and the matrix was then washed with 50 mM sodium citrate, 1 M NaCI, 0.1 M arginine x HCI, pH The column was then washed again with the equilibration buffer, after which elution with 0.1 M glycine, pH 2.5, followed. The eluate (about 30 ml) was ::collected in a volume of 3 ml of a 200 mM sodium citrate solution, pH 5.5, with stirring and then adjusted to a pH of SThe eluate solution was used for further analysis. An SDS-PAGE with subsequent transfer to a PVDF membrane and detection of the factor VII activator band was carried out using the unreduced and using the reduced sample. Activity tests of the proteins thus obtained were carried out according to the process described in German patent application 199 26 531.3, namely the activation of prourokinase and factor VII, with subsequent detection of urokinase or activated factor VII. The amounts of protease tested in this system, determined as protease antigen, correspond to the expected theoretical activity, whereby the activity of the isolated protease or of the proenzyme with respect to the biological activity was shown.

12 Example 2 Anion-exchange chromatography A solution containing the proenzyme form of the factor VII-activating protease which still contained contaminations by other proteins was pumped onto a Mono Q Sepharose in a buffer solution of 20 mM Na acetate, 0.1 M glycine, pH and then washed with the abovementioned buffer. The fraction passing through was collected. Bound proteins were eluted using 20 mM Na acetate, 2 M NaCI, pH 4.5. The eluate was diluted in a buffer of 5 mM Na citrate, 50 mM NaCI, pH and investigated in the test systems mentioned in Example 1. Aliquots were stored at 4 to 8°C or frozen at -200C.

After storage of the eluate solution at 6°C for several days, the tests were repeated, the dilutions of the (thawed) samples in each case being carried out shortly before the test.

SDS-PAGEs/Western blots confirmed that the protease had been isolated in its proenzyme form. After SDS-PAGE and staining of proteins by means of Coomassie Blue, in addition to the protease a number of contaminating proteins, which were also to be found in the fractions flowing through, were visible in the starting solution (before chromatography). The protease was represented as a band corresponding to the proenzyme form even after reduction) in pure form. The activity tests (see Example 1) confirmed the nativity of the protein in the sense of the retainment of the biological activities.

Claims (18)

1. A process for the preparation in pure form of at least one of the protease activating blood clotting factor VII or its proenzyme from biological fluids or genetically engineered preparations: a) by at least one of anion- or cation-exchange chromatography at a pH below the isoelectric point of the protein to be isolated, or b) by at least one of: i) a combination of anion- or cation-exchange chromatography with affinity chromatography, or ii) fractional precipitation at pHs of between 2.5 and the affinity-chromatography separation processes being carried out either using: calcium phosphate/hydroxyapatite, and/or a hydrophobic matrix, and/or a chelate matrix, 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; wherein the protease activating blood clotting factor VII: a) is inhibited by the presence of aprotinin, b) is increased in its activity by calcium ions and/or heparin or heparin- related substances and c) in SDS-PAGE, on subsequent staining in the non-reduced state, has one or more bands in the molecular weight range from 50 to 75 kDa and in the reduced state has a band at 40 to 55 kDa and one or more bands in the molecular weight range from 10 to 35 kDa.

2. The process as claimed in claim 1, wherein the fractional precipitation is conducted at pHs of between 2.5 and 7.2.

3. The process as claimed in claim 1, wherein the fractional precipitation is conducted at pHs of between 2.5 and COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21 21/04 '05 THU 11:44 FAX 61 2 9888 7600 WATERMARK (1013 14

4. The process as claimed in any one of claims 1 to 3, carried out in the presence of one or more protein stabilisers which are selected from the group consisting of: complexing agents of divalent ions, divalent ions, amino acids, sugars, alcohols solubilisers, detergents, proteins, reductants, and proteinase inhibitors or synthetic inhibitors.

5. The process as claimed in any one of claims 1 to 4, wherein, for the chromatographic separation of the protease activating factor VII from its proenzyme by means of stepwise elution, a substance is immobilized on the support material which has bonds of different strength to the protease on the one hand and to the proenzyme on the other hand, and the different eluates are then collected separately from one another and the respective protein is isolated from them.

6. The process as claimed in any one of claims 1 to 5, wherein the fractional precipitation of the at least one protease or its proenzyme from its solution is carried out by addition of polyethylene glycol from a concentration of at least 10% by weight,or ammonium sulfate from a concentration of at least 15% by weight.

7. Use of at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme together with one or more of the protein stabilisers as set forth in the process of claim 4, in the manufacture of a medicament lor one or more of the following purposes: COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21 21/04 '05 THU 11:44 FAX 61 2 9888 7600 WATERMARK li014 for assisting blood clotting in the case of a tendency to bleeding, in the case of a lack of factors of the intrinsic clotting pathway or if factors of the intrinsic clotting pathway are blocked by inhibitory antibodies, for the prophylaxis and/or therapy of syndromes associated with thrombotic complications, for the prophylaxis and/or therapy of inherited or acquired deficiency states of the protease or of its proenzyme, and for assisting wound healing either alone or as a constituent of a fibrin adhesive or a fibrin fleece or patch, wherein the medicament is optionally in combination with growth factors, administered either subcutaneously, intramuscularly, intradermally or topically. .4.

8. A method for assisting blood clotting in a patient having a condition *selected from: having a tendency to bleed, having a lack of factors of the intrinsic clotting pathway and, having factor VIII inhibitor bypassing activity, which method i..ncludes administering to the patient a therapeutically effective amount of at least *one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme. 4

9. A method for the prophylaxis and/or therapy of condition(s) selected from: syndrome(s) associated with thrombotic complications, and inherited and acquired deficiency states of the protease activating blood clotting factor VII or its proenzyme, which method Includes administering to the patient a therapeutically effective amount of at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme. A method for assisting wound healing in a patient, which method includes administering to the patient a therapeutically effective amount of at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme.

COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21 21/04 '05 THU 11:44 FAX 81 2 9888 7800 WATERMARK (a015 16

11. The method as claimed in claim 9 or claim 10 wherein the manner of administration is selected from: topical, subcutaneous, intramuscular and intradermal administration.

12. The method as claimed in claim 10 wherein the protease activating blood clotting factor VII or its proenzyme is administered in the form of a constituent of a fibrin adhesive or bound to a suitable carrier matrix, optionally in combination with growth factors.

13. The method as claimed In claim 12 wherein the suitable carrier matrix is in the form of a fleece or of a patch.

14. Use of at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme together with one or more of the protein stabilisers as set forth in the process of claim 4, in the manufacture of a pharmaceutical preparation for the coating of surfaces of articles consisting of plastic or metals to be implanted in the body.

The use of claim 14 wherein the articles to be implanted in the body are selected from synthetic heart valves, blood vessels and cannulas inserted for taking blood or for artificial feeding.

16. A reagent comprising at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme together with one or more protein stabilisers as set forth in the process of claim 4 when used in biological test systems and for antigen detection.

17. A process for the preparation in pure form of at least one of the protease activating blood clotting factor VII, as defined in the process of claim 1, or its proenzyme, which process is substantially as hereinbefore described with reference to Example 1. COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21 21/04 '05 THU 11:44 FAX 61 2 9888 7600 WATERMARK 0~016 17

18. Protease activating blood clotting factor VII, as defined in the procesS of claim 1, or its proenzyme prepared by the process of any one of claims 1 to 6 or 17. DATED this 21 st day of April 2005 ZLB BEHRING GMBH WATERMARK PATENT TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P1 7905AUOO 00 -:9 .0 COMS ID No: SBMI-01214914 Received by IP Australia: Time 11:49 Date 2005-04-21