DE2440254A1 - INSOLUBLE ENZYME PREPARATION AND METHOD FOR MANUFACTURING IT - Google Patents

Description

PENTAPHARM A.G., Basel

Insoluble enzyme preparation and process for its manufacture

The present. Invention relates to an insoluble enzyme preparation that is used in blood tests and in the recovery finds use of coagulation factors from human or mammalian blood. The invention also relates to a method for the production of the named enzyme preparation.

The removal of fibrinogen from blood, plasma and its fractions proves itself in the preparative isolation as well as in the analytical determination of individual blood coagulation factors as necessary.

Fibrinogen impairs the solubility and shelf life of injectable concentrates of coagulation factors intended for human medical purposes, and is an impurity that is difficult to remove in the pure production of factor VIII, factor XIII, prothrombin, plasminogen, etc. [see aB EF MAJiMEN in "Thrombosis and Bleeding Disorders "

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by N. BANG et al., p. 140, Stuttgart / New York / London (1971)]. Fibrin formed from fibrinogen adsorbs and inhibits thrombin. The removal of fibrinogen from plasma is therefore always important in the quantitative determination of blood coagulation factors necessary if their activity is measured via the activation of prothrombin or via the inhibition of thrombin.

L. FREDERICQ [Annales Soc. med. Gand, 55, 93 (1877)] first described the separation of fibrinogen from plasma samples by heating them to 56 ° C. This method, which is still used today, has the disadvantage that antithrombin III and prothrombin are partially destroyed by heating [see, for example, BLOMBACK, M. BLOMBACK and P. OLSSON, Thromb. Diath. Haem., 9 ,, 368 (1963)], so that this method leads to errors in its analytical application. Since the heating to 56 ° G may only last 3-5 minutes, this method cannot be used for the preparative extraction of blood coagulation factors, where the heating and cooling time of large plasma volumes is long. Defibrinogenation by adding small amounts of thrombin, which is also commonly used, is also inadequate in analysis because excess thrombin on the one hand simulates too high an activity in the prothrombin determination and on the other hand because antithrombin is consumed by thrombin in the antitirombin determination and thus analysis values that are too low are achieved [JM GREEP et al., Thromb. Diath. Haem., 5_, 256 (19S0)].

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Thrombin also causes molecular changes in the blood coagulation factors V, VIII and XIII [see S.P. COLOWICK & N.O. KAPLAN, "Methods in Enzymology", Vol. XIX, Proteolytic Enzymes, Academic Press, New York and London, pp. (1970)]. It is therefore not suitable for preparative defibrinogenation of plasma and its fractions. Removal of fibrinogen from plasma by adsorption on bentonite has the disadvantage that the factors V and X are also adsorbed [see M.P. ESNOUF & K. LECHNER in: N. BANG, F. BELLER, E. DEUTSCH & E. MANNEN, "Thrombosis and Bleeding Disorders", Stuttgart, New York, London, p. 176 (1971)].

P.W. HOWIE et al. [Brit. Journ. of Hematology, 2J5, 101 (1973)] defibrinogenized plasma for antithrombin III determination with ancrod, the throrabin-like enzyme from agkistrodon

rhodostoma venom. Compared to thrombin, this enzyme offers the advantage of not consuming any antithrombin and, furthermore, not activating to act on Factors VIII and XIII. But it has the disadvantage that it proteolytic the alpha chain of fibrin degrades [P. MTTOCK & M.P. ESNOUF, Nature New Biology, 233, (1971)] and thereby a thrombin inhibition by fibrin cleavage products can cause. In addition, an excess of ancrod remaining in the plasma must be replaced by a specific antiserum be neutralized, which also contains antithrombin and its use this defibrinogenation method

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much more expensive. S. LOPACIUK [VIII Congr. World Fed. of Haemophilia, July 24, 27, Buenos Aires (1972)] describes a Method for removing fibrinogen from factor VIII concentrate by incubating it with batroxobin, the thrombin-like one Enzyme from Bothrops atrox venom. This enzyme offers the same advantages over thrombin as ancrod has over the latter, however, has the additional advantage of not causing any proteolytic degradation of the alpha chain of fibrin. Another disadvantage of using Batroxobin is the fact that excess enzyme remains in the preparation and must not be neutralized by an antiserum, as this would contaminate the for parenteral administration certain preparations would be made by foreign protein on humans.

It has de found that one can get the cons of the above can eliminate defibrinogenizing rivulets described by using thrombin-like snake venom enzymes which have been made insoluble by binding to insoluble carrier substances and thereby easily separate from blood plasma permit.

The invention relates to an insoluble enzyme preparation, which is intended for blood tests and for the extraction of blood clotting factors from human

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or mammalian blood, blood, plasma, or fractions thereof to be added in order to remove the fibrinogen and which is characterized by the fact that it is an insoluble Contains carrier substance to which a thrombin-like, defibrinogenic active enzyme from snake venom is bound.

Under "insoluble" is insolubility in water, aqueous solutions of salts, and dilute mineral acids organic acids, dilute aqueous bases, organic solvents, blood plasma and fractions thereof, blood serum, etc. to understand.

The insoluble ena ^ n preparation is according to the invention

obtained by having a thrombin-like, defibrinogenic active enzyme from snake venom with a carrier substance containing reactive groups converts its reactive groups with functional groups of the enzyme are able to react to form an insoluble reaction product.

Suitable carrier substances are generally substances which may have been activated beforehand and which have the property of being proteins, in particular serine proteases [see KA WALSH & PEWILCOX in "Methods of Enzymology", Vol. XIX, p. 31 ', SP COLOWICK &. NO KAPLAN, Academic Press, New York and London, (1970)] in such a way that the proteins mentioned become insoluble without their biological activity being destroyed.

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Such methods for insolubilizing proteins are known per se. Applying these methods to However, snake venom enzymes have not yet been described.

Insoluble polymeric substances that are activated by introducing reactive groups or by catalysis are suitable as carrier substances. Cellulose [DH CAMPBELL et al, Proc. nat. Acad. May be. USA, 31, 575 (195I) J AE GURVIC et al., Biokhimiya 24, 144 (1959) i AE TUMANOVA, Biokhimiya 26, 803 (1961), N. GRUBHOFER, Hoppe Seylers Z. physiol. Chem. 297, 108 (1954); MA MITZ et al., Nature 189: 576 (1961); CJ. EPSTEIN et al., J. biol. Chem., 237, 2175 (1962)], polydextrans [J. PARATH, Nature, 183 . 1657 (1959); J. PARATH, Nature, 218, 834 (1968)], agarose [J. PARATH, Nature, 218: 834 (1968); R. AXEN et al., Nature, 214 . 1302 (1967); P. CUATRECASAS et al., Biochem. III, 2291 (1972); P. CUATRECASAS et al., J. biol Chem. 245, 3059 (1970)], Polypeptides [A. BAR-ELI et al., Nature 18 £, 856 (1960); E. RIESEL et al., J. biol. Chem., 239, 1521 (1964); D. GIVOL et al., Biochem. 3, 444 (1964)], polystyrenes [G. MANECKE et al., Makromol. Chem., 39, 13 (1960); G. MANECKE et al., Makromol. Chem., 51, 199 (1962); G. MANECKE et al., Makromol. Chem., 91, 136 (1966)], polyacrylic acid !! [JP CORNAZ et al., HeIv. chim. Acta, 40, 2015 (1957); K. MOSBACH, Acta chem. Scand., 24, 2084 (1970)], polyacrylamides [K. MOSBACH, Acta ι formerly Scand. 24, 2084 (1970)] and copolymers [K. KOSBACH, Acta chem.

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Scand., 24, 2084 (1970); SA BARKER et al. *, Carbohyd. Res., 14_, 287 (1970); G. MANECKE et al., J. Polymer. May be. Part C, 30 , 607 (1970)]. The reactive groups introduced can, for example, be diazonium groups [DH CAMPBELL et al., Proc. nat. Acad. May be. USA, 37, 575 (1951); AE GURVIC et al., Biokhimiya 24, 144 (1959); JI CEBRA et al., J. biol. Chem., 236., 1720 (1961)], isocyanate groups [H. BRANDENBERGER, HeIv. chim. Acta 40, 61 (1957); R. AXEN et al., Nature 210, 367 (1966)], acid chloride groups [HC ISLIKER, Advanc. Protein Chem., 12., 387 (1957)], azido groups [MA MITZ et al., Nature 189 . 576 (1961); CJ. EPSTEIN et al., J. biol. Chem. 237, 2175 (1962)], acid anhydride groups [T. LEWIN et al. » Bio chem. 3_, 1905 (1964)], SuIfοChloridgruppen [HC ISLIKER, Advanc. Protein Chem., 12, 387 (1957)], dinitrofluorophenyl groups [G. MANECKE et al., Makromol. Chem., 39, 13 (1960) J G. MA-NECKE, Naturwissenschaften, 51, 25 (1964)], isothiocyanate groups [R. AXEN et al., Nature 210: 367 (1966); G. MANECKE et al., Naturwissenschaften, 54, 531 (1967)], triazine halide groups [G. KAY et al., Nature 216, 514 (1967)], hydroxy groups [R. AXEN et al., Nature 214: 1302 (1967); P. CUATRECASAS et al., J. Biol. Chem., 245_, 3059 (1970)], amino groups [DH CAMPBELL et al., Proc. nat. Acad. May be. USA 37: 575 (1951); AE GURVIC et al., Biokhimiya 24 »1 * 4 (1959) j P. CUATRECASAS et al., J. Biol. Chem., 245, 3059 (1970)], N-hydroxysuccinic acid imide ester groups [P. CUATRECASAS et al.,

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Biochem. 11, 2291 (1972)] of the hydrazino groups [F. MICHAEL et al., Makromol. Chem. 3., 200 (1949)]. There will be

Carrier substances used moderately, their reactive poison

ness binding of Schlanger / enzymes under mild conditions as possible ermogli cht ·.

As thrombin-like, defibrinogenic Enzymes can be used, for example, the enzymes obtainable from the venom of the snakes listed below:

CROTALIDAE ;

Genus Agkistrodon, e.g. the species acutus, rhodostoma, and others;

Genus Bothrops, e.g. the species atrox, jararaca, moojeni, marajo ^ ensis, asper, pradoi, and others;

Genus Crotalus, e.g. the species adamanteus, atrox, durissus terrificus, and others;

Genus Trimeresurus, for example the species flavoviridis; VIPERIDAE:

Genus Vipera, for example the species aspis, berus, etc.; ELAPIDAE:

e.g. the species Denisonia superba, Notechis scutatus, Pseudechis porphyriacus.

Particularly favorable results are achieved if the thrombin-like snake venom enzyme Batroxobin, the enzyme obtainable from the poison of Bothrops atrox is used. The properties of this enzyme and how to obtain it are described, for example, in Belgian Patent No. 778.226.

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The reaction of the enzyme with the active carrier substance is expediently in an aqueous medium,

e.g. in dilute aqueous buffers such as aqueous sodium buffers bicarbonate, phosphate buffer or borates, in the neutral to weakly alkaline range, and at low temperatures, e.g. in the range from about 0 ° to room temperature. The resulting insoluble enzyme preparation can by washing with aqueous buffer or salt solutions of enzyme that is still adhering, but not chemically bound, can be released. It is advisable to work on the carrier substance of the enzyme preparation obtained to inactivate any free reactive groups still present. The inactivation can by treatment with inactivating substances, e.g. ß-naphthol, amino acids, weak alkalis or weak Acids are brought about.

If the enzyme preparation according to the invention is human or mammalian blood or blood plasma or fractions thereof added, then defibrinogenation of the blood or plasma takes place through the formation and precipitation of "fibrin, which may be is reflected on the insoluble enzyme preparation. The precipitated fibrin and the enzyme preparation or that with Fibrin-laden enzyme preparations can, for example, be separated by pressing and centrifuging.

The enzyme preparation which has been separated off and optionally loaded with fibrin can be regenerated by e.g.

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2AA0254

- ίο -

5 molar aqueous urea solution treated, whereby the coagulated fibrin goes into solution. The enzyme preparation freed from fibrin is expediently treated with aqueous NaCl (e.g. 0.9 # ig) and is then for reuse ready.

The weight ratio between the enzyme and the carrier substance in the enzyme preparation according to the invention can vary within a broad range. Favorable results have been obtained with a weight ratio of about Scored 1 in 20.

In the following exemplary embodiments, temperatures are given in degrees Celsius.

example 1

100 mg of p-aminobenzyl cellulose were in an ice bath suspended with stirring in 2-10 ml of 2N HCl. 4% aqueous NaNOp was added to the suspension and allowed to react for 15 minutes. The cellulose benzyldiazonium hydrochloride formed was then sucked removed the excess hydrochloric acid by washing with cold water and suspended the residue in 2-4 ml of 0.1 molar Phosphate buffer of pH 7.5. 5 mg of batroxobin were dissolved in 1 ml of the same buffer and the solution was stirred during 24 hours at 0 to 5 °. The reaction product was sucked off, washed with water and then with a saturated

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Solution of β-naphthol in aqueous sodium acetate for 12 hours treated at 0-5 °. The enzyme preparation obtained was then thoroughly washed several times with 0.1 molar phosphate buffer of pH 7.5 washed with 0.5 molar NaCl and with 0.9 # NaCl solution, until no more enzyme could be detected in the wash water.

The formation of the enzyme preparation according to Example 1 can be illustrated by the reaction scheme below will:

Cellulose-O-CH ₂ -

Cellulose-O-CHg-Cellulose-O-CHg-

-NH,

ENT, HCl

-N = N ⁺ Cl "+

enzyme

COOH

Example 2

A solution of 300 mg of BrCN in 4 ml of water was adjusted to pH 11.5 with 2N NaOH while stirring. In addition The mixture was given 100 mg of polydextran (branded product Sephadex) in 4 ml of v / ater and the pH was adjusted to 11.5 with 2N NaOH. After 10 minutes, the reaction product was cut off and washed with cold water. 5 mg of batroxobin were dissolved in 1 ml 0.5 molar sodium bicarbonate solution, the solution was added. to the

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activated polydextran gel and stirred the reaction mixture at 0-5 ° for 12 hours, after which the was obtained Enzyme preparation with bicarbonate solution, dilute hydrochloric acid, 1 molar NaCl and water washed until there is no enzyme in the wash water

will
more proven / could.

The formation of the enzyme preparation according to Example 2 can be illustrated by the following reaction scheme:

Polydextran-OH + NaOH + CNBr > ·

[Polydextran-CN intermediate] + -NH,

enzyme

₂ -COOH

Polydeacbran-0-CH = N-

enzyme

-COOH

Example 3

Hydrazines / '

100 mg of polyacrylamide resin (branded product Enzacryl AH) were suspended in 10 ml of 2N HCl and stirred in an ice bath. 4 # strength sodium nitrite solution was added to the suspension and the reaction mixture was allowed to react for 15 minutes. The activated polyacrylamide resin obtained was then centrifuged off and washed neutral with 0.1 molar phosphate buffer. 5 mg of batroxobin were dissolved in 1 ml of phosphate buffer and cooled in a fiis bath. the

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Solution was added to the activated polyacrylamide resin with stirring. The reaction mixture was left at 0-5 ° for 24 hours react, centrifuged the enzyme preparation obtained and washed it several times with phosphate buffer, 0.5 molar NaCl solution, Water and 0.9% saline solution until the wash water is free of enzyme was.

The formation of the enzyme preparation according to Example 3 can be illustrated by the reaction scheme below will.

[-CH ₂ -CH-CH ₂ -CH-CH ,, -

CONH,

ι d. \ - «n CO-NH-NH ,,

ENT,

[-CH ₂ -CH-CH ₂ -CH-CH ₂ V] _n + H, CO-NH ₂ CO-N ₃

enzyme

WH,

COOH

[-CH ₀ -CH-CH ₀ -CH-CH ₀ -C {1

· ₂ - _τ » ₂ -w ₂ - _ ^, _n

JO CO-NH ₂ ^{ 2nd

enzyme

■ COOH

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The following examples show how the enzyme preparation according to the invention is used to defibrinogenize blood plasma can be used.

Example 4

0.2 ml of citrated plasma was mixed with 0.1 ml of a suspension of the enzyme preparation obtained in Example 3 (100 mg enzyme preparation per ml) mixed in 0.9 # NaCl solution. The mixture was constantly shaken. After 5-10 minutes, FibrinfSden could be observed on the particles of the enzyme preparation had knocked down. After 10-15 minutes, the fibrin condensed into flakes. After about 1/2 to 1 hour the clot was squeezed out and the defibrinated plasma was centrifuged off. The defibrinated plasma coagulated when added from fresh enzyme preparation or from thrombin and was therefore no longer required for further hematological investigations, such as Antithrombin and prpthrombin determinations, use-related

Example 5

Factor VIII was obtained from 10 liters of citrate-containing bovine plasma according to the method of JG POOL et al. [Nature, 203 . 312 (1964)] separated by precipitation at 0 to + 2 ° and centrifugation. The cryoprecipitate which costs about $ 60

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consisted of fibrinogen, was dissolved in 200 ml of 0.05 molar ammonium carbonate solution dissolved at 20-25 °. 2 g of the insoluble enzyme preparation prepared according to Example 3 were added to the solution added. The mixture was stirred at 20-25 ° for 1 hour. The fibrin precipitate formed and the suspended insoluble one Enzyme preparations were separated by centrifugation at 3000 revolutions per minute for 15 minutes. The centrifigate was cooled to -3 ° to 0 ° and mixed with 35 ml of cooled (0 °) ethanol and then with 15 g of glycocolla. the The mixture was stirred for 1-3 hours at -3 ° to 0 °, whereupon the flocculated factor VIII was separated off by centrifugation became. The sediment was dissolved in 0.055 molar trinatriuric citrate solution solved. The solution was centrifuged and sterile filtered through a membrane filter. 6000 bis were obtained 8000 units of a readily soluble, fibrinogen-free factor VIII preparation.

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Claims (5)

24A0254 CLAIMS 1. Insoluble enzyme preparation that is used in blood tests and in the extraction of blood clotting factors from human or mammalian blood to be added to the blood, plasma or fractions thereof, to remove the fibrinogen, characterized in that it contains an insoluble carrier substance to which a thrombin-like, defibrinogenic enzyme Snake venom is bound. 2. A process for the production of an insoluble enzyme preparation which is intended to be added to the blood plasma or fractions of the same in blood tests and in the production of blood coagulation factors from human or mammalian blood in order to remove the fibrinogen, characterized in that a thrombin-like , defibrinogenic ^ end acting enzyme from snake venom is reacted with a carrier substance having reactive groups, the reactive groups of which are able to react with functional groups of the enzyme to form an insoluble reaction product. 3. The method according to claim 2, characterized in that that one as a carrier substance «one in advance if necessary 509810/1124 activated polymeric substance used, which has the property of dissolving soluble proteins, in particular serine proteases, to bind in such a way that the proteins mentioned become insoluble without their biological activity being destroyed. 4. The method according to claim 2 and 3, characterized in that activated cellulose, activated polydextrans, activated agarose, activated as a carrier Polypeptides, activated polystyrenes, activated polyacrylic acids, activated polyacrylamides or activated copolymers used. 5. The method according to claim 2 and 3 :, characterized in, that one uses the enzyme batroxobin. 509810/1 124