DE1617681B2 - PROCESS FOR OBTAINING AN ANTICOAGULATING AND DEFIBRINATING ENZYME WITH ANTICOAGULATING AND DEFIBRINATING ENZYME FROM THE POISON OF THE VIPER ANCISTRODON RHODOSTOMA - Google Patents

Description

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The subject of the main patent is an in vivo anticoagulant and defibrinating enzyme from the venom of the viper Ancistrodon Rhodostoma, which is characterized in that a) its biological activity is thrombin-like, ie it produces fibrin with a bundle-like character, but that it does not have a proteolytic effect in vivo and that its effect in vivo is quickly reversed by means of a specific antiserum against the poison of the viper Ancistrodon Rhodostoma, b) it is adsorbed on di- or triethylaminoethyl cellulose, c) it is soluble in physiological saline solution, d) it has an electrophoretic mobility of 3, 92 · 10 ⁵ V / cm · lake mol in 0.1. Has phosphate buffer at pH 7.0, e) it has a molecular weight, when determined in the ultracentrifuge, of about 000 to about 40,000, f) it has a sedimentation coefficient S20W = 3.40 Svedberg, g) it has a partial specific volume of 0.66, h) it shows two Precipitat bands in the immunoelectrophoresis against a specific antiserum against the venom of the viper Ancistrodon Rhodostoma by the agar diffusion method, i) its biological activity through diisopropyl fluorophosphate in a concentration of ΙΟ ^-3 Mol / l at pH 8 and room temperature is not inhibited within 5 minutes and j) that it is protein-like and largely colorless in the pure state. This enzyme can be obtained according to claim 2 of the main patent by chromatographing the poison of the viper Ancistrodon Rhodostoma on a column of a weakly basic anion exchanger material and collecting the fraction containing the active material largely free of undesired proteolytic enzymes and isolating the enzyme from it. In this purification carried out by chromatography and suitable weakly basic anion exchangers those which contain a tertiary amino group, e.g. B. triethylaminoethyl cellulose.

It has now been shown that as a result of fluctuations in the properties of the commercial product of Approach to approach, the triethylaminoethylcellulose not in every case according to the one in the main patent, In particular, the method described in Example 1, a sufficiently clear separation between the Enzyme-containing fraction and the fraction immediately preceding this results. As a result is the purified product obtained by this procedure described often still with a low Part of a hemorrhagic factor present in the poison is contaminated, the greater part of which goes into the previous elution steps has been eliminated. Although the contamination is not so great that it is at The dose considered of the active substance to be administered can be dangerous, it is however, it is desirable to obtain as pure a product as possible, especially when the product is to be administered by intramuscular or subcutaneous injection. The required degree of purity can by suitably varying the molarities of the eluents in the respective stages of the purification process be achieved. Unfortunately, however, there is a change in the elaborated procedure to a reduction in the yield of active ingredient.

In a further embodiment of the method according to claim 2 of the main patent 14 42134 has now been found that the above-mentioned impurity, i.e. H. the hemorrhagic factor, largely eliminated can be if, as stated in claim 1, the eluate of the exchange column or the Subjected raw poison to a further purification stage using gel filtration.

Gel filtration is a mode of operation that uses materials that are commonly known referred to as "molecular sieves". These substances are insoluble, practically inert products with the property that they separate molecules on the basis of their permeability through those with the solvent swollen substances allow a property that is related to the molecular size and the Shape of the dissolved molecules. These molecular sieves are manufactured by various companies and each is characterized by an approximate useful molecular weight range, within the same fractionated the material. It is important to choose the most suitable material so that a good separation of the thrombin-like active substance

and the hemorrhagic factor. Typical suitable substances are cross-linked dextrans, Polyacrylamide gels and galactose polymers. These materials fractionate well in the molecular weight range from 20,000 to 100,000.

The advantage of gel filtration is that the fractionation takes place in a wide range of buffer systems can be made with the separated components in the chosen eluent are preserved and free from the buffer system of the original impure preparation, that the column was given up.

Thus, according to the main patent in the fractionation with a triethylaminoethyl cellulose column fraction 6 obtained the 0.1m tris (hydroxymethyl) aminomethane / phosphate Contains buffer (tris / phosphate) of pH 6, with cross-linked! Dextran on a background of 0.1m sodium phosphate, 0.1m NaCl of pH 7.0, the separated thrombin-like agent is obtained in the latter solvent and is free from the Tris / phosphate buffer. Any suitable aqueous base solvent can be selected; it is however, it is desirable to choose a system of adequate activity stability that is sufficiently high Having ionic strength to eliminate abnormal absorption effects associated with some proteins e.g. B. in pure Water can act as the basic solvent. Approximately isotonic solutions such as B. the above In the present case, common salt / phosphate buffers are free from such effects.

Since the fractionations by gel filtration on a given gel are primarily a function of the size of the column and the volume applied, it is desirable to reduce the latter to as small a volume as possible in order to achieve the greatest possible separation. For this purpose, triethylaminoethyl cellulose eluates are expediently concentrated ^{at about 30 ° C. under reduced pressure.} It can be concentrated to a concentration of 3 m Tris / phosphate, ie up to 30 times the 0.1 m Tris / phosphate buffer at pH 6 without loss of activity. The concentrated eluate can then be fed to a column of suitable size. The high ionic strength of the liquid applied does not appear to be a disadvantage. Some common methods of concentration such as ultrafiltration, on the other hand, are associated with substantial losses of thrombin-like activity.

The invention is explained in more detail using the following example:

example

Fractionation of raw poison with
Triethylaminoethyl cellulose

The fractionation was carried out according to the procedure of the main patent. On the elution of the Proteins were followed by a determination of the UV absorption with a continuously operating device 254 πιμ and the measurements of the individual fractions at 280 Γημ.

2 g of the crude poison were dissolved in 70 cm ^{3 of} 0.01 m Tris / phosphate with a pH of 8.5 and the insoluble white sediment was thrown off. The clear yellow supernatant liquid was added to the triethylaminoethyl cellulose column (35 cm × 3.9 cm). The nominal ion exchange capacity of 0.65 meq / g was treated and the column was loaded according to the procedure previously used. After loading, the column was eluted with the same solvent, ie with 0.01 M tris / phosphate with a pH of 8.5.

The complete scheme of the elution carried out at room temperature for 2 days at 250 cm ³ / h is given below. The approximate volume of the eluent is listed, but the actual change in eluent is determined by reading the UV absorption.

Eluent 1 0.01 m Tris / phosphate, pH 8.5

Eluent 2 0.01 m Tris / phosphate, pH 7

Eluent 3 0.02 m Tris / phosphate, pH value 6

Eluent 4 0.06 m Tris / phosphate, pH 6

Eluent 5 0.1 M NaCl, 0.09 M Tris / phosphate, pH value

Eluent 6 2 m NaCl ₁ 0.09 m Tris / phosphate, pH value

750 cm ³ fraction I, II and III 2000 cm ³ " fraction IV 3000 cm ³ fraction V 1500 cm ³ fraction VI 500 cm ³ fraction VII 1000 cm ³ fraction VIII

Fractions I and II were not separated as well as the earlier ones described in the main patent Fractionations. Fraction III is larger, presumably from the earlier fraction IV, which is now very much is much smaller, protein gets into this fraction. The change in the new system with the eluents 1 and 2 (these eluants are identical to those used in the earlier experiments) is true corresponds to proteins that are eluted at a low ionic strength. Washing out between the Fractions IV and V were now omitted, and fraction V, which had previously been dated with 0.04 m Tris / phosphate pH 6 had been eluted, it was now eluted with 0.02 M buffer solution of the same pH value, the Protein appeared in three flat maxima over a longer period of time. The thrombin-like activity, measured as described in the main patent, gradually increased in these fractions. Group VI was named a same maximum elutes as before (with 0.09 m Tris / phosphate of pH 6) with an initial one Wetting the protein and an increase in thrombin-like activity, followed by a gradual or gradual decline. Fractions VII and VIII were eluted as before with similar protein profiles, as is the case with the large differences in ionic strength compared to the fraction V eluent was expected.

Most of the yellow-colored material of the raw poison was eluted in fraction VII. A minor one However, the portion of the colored solution appears in fraction VI, after the main protein and the Maximum thrombin-like activity, but significant amounts of activity still eluting will. From measurements of the optical density at 280 πιμ and 440 πιμ with the colored fractions as well by comparison with similar measurements on the colored fraction VII (containing 1-amino acid oxidase Material) it can be concluded that the colored total fraction of fraction VI is not protein-bound is and probably a breakdown product of flavoproteins originally contained in the raw poison is.

Fractionation of Fraction VI on
network! Dextran

The collected material of fraction VI, which was obtained from 2 g of raw snake venom and up to 1200 cm ³ containing 70 to 80% of the original thrombin-like activity of the raw venom, was concentrated on a roller evaporator at 30 ° C / 20 mmHg. It was concentrated up to 50 times, without any practical loss of activity, despite the high molarity of the tris / phosphate buffer (up to 3m) achieved in the process.

650 cm ³ of a collected fraction VI preparation with the activity of 97 Thrombineinheiten per cm ³ was concentrated 20 times at 3O ⁰ C. Virtually all of the concentrate (32 cm ³ ) was placed in a 97 cm χ 6 cm column filled with crosslinked dextran and containing 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.0, containing 0 , 1% chloroform, had been equilibrated. Washing was continued with the same solvent at room temperature at 80 cm ³ / h and fractions of 8 cm ^{3 were} collected. The absorption at 280 μm was determined in each case and the thrombin-like activity was then checked. The activity in thrombin ^{units per cm 3} divided by the optical density at 280 ΐημ gives a measure of the strength.

The maximum of the thrombin-like activity is strongest at an elution ^{volume of about 990 cm 3} and the maximum of the hemorrhagic factor at a volume of 1290 cm ³ with good separation between the two. The fractions between 952 cm ³ and 1075 cm ³ , which have specific activities (activities in thrombin units per cm ³ divided by the optical density in a 1 cm cell at 280 ιτιμ) better than 800, are combined. The resulting mixture had an optical Density at 280 πιμ of 0.33 and the control gave 320 thrombin units per cm ³ . This corresponds to a recovery of purified thrombin-like active ingredient from fraction VI of 71%, as well as an overall recovery from the crude product of 50 to 55%.

Haemorrhagic attempts against a standard preparation indicated a hemorrhagic factor content of about 0.5% on the basis of a Absorption at 280 ηιμ.

The yellow-colored component of the Fraction VI solution, which after concentrating before loading the Column is clearly visible, is strongly retained on crosslinked dextran, since it is used as a material fairly low molecular weight migrates. It is well recognized by both the peak of thrombin-like activity and the peak of the hemorrhagic factor.

The finally collected solution with thrombin-like activity was diluted in physiological saline solution to an activity of about 70 thrombin units per cm ³ and sterilized by membrane filtration. The sterile solution was filled into standard 1 cm ³ all-glass ampoules in volumes of 1.1 cm ^{3 each and sealed.} This makes suitable single dose levels for intravenous or intramuscular injection.

Claims (3)

Patent claims: 1. A method for obtaining the enzyme according to claim 1 of patent 14 42 134, wherein one after Claim 2 of this patent the poison of the viper Ancistrodon Rhodostoma on a column of a weakly basic anion exchange material and chromatographed the active material containing fraction largely free of undesired proteolytic enzymes and the enzyme isolated therefrom, characterized in that that (A) you first put the raw poison on a column of a weakly basic, tertiary Amino group-containing anion exchanger adsorbed and then increasing with buffer solutions Molarity from pH 5.5 to 7.5 first the unwanted proteolytic enzymes and then the desired enzyme is fractionally eluted by the eluate thus obtained and containing the enzyme gel filtration on a material selected to allow good separation of the enzyme and the hemorrhagic factor is achieved, and that one finally submits the enzyme in is isolated in a known manner from the eluate of the gel filtration or that (B) the gel filtration is carried out first of the raw poison on a material which is selected so that a good separation of the enzyme and the hemorrhagic factor is achieved, carries out that one then the thus obtained the enzyme containing fraction of the gel filtration step on a column of a weakly basic, tertiary Amino group-containing anion exchanger adsorbed and then increasing with buffer solutions Molarity from pH 5.5 to 7.5 first the unwanted proteolytic enzymes and then the desired enzyme is eluted fractionally, and that finally the enzyme from the eluate of the Anion exchange treatment wins in a known manner. 2. The method according to claim 1 (A), characterized in that the enzyme containing The eluate of the anion exchanger is concentrated and the gel filtration is then carried out. 3. The method according to claim 1 or 2, characterized in that the material for the Gel filtration uses a crosslinked dextran.