NO135709B - - Google Patents

Description

The invention relates to a method for cleaning

and crystallization of kallikrein, which. is a mixture consisting of the components kallikrein A and kallikrein B, and production of the possibly crystalline components A and B.

By influencing the body's own kininogen, kallikrein releases the circulating kinins. The kallikrein preparations are therefore used therapeutically for the therapy of various bleeding disorders (E.K. Frey, H. Kraut, E. Werle, Das Kallikrein-Kinin-System und seine Inhibitoren, F. Enke, Stutt-

gart 1968, page 150 et seq.).

Until now, partially purified preparations were used for this

rates of kallikrein by pancreas, Submaxillaris or urine.

It is already known that it is possible to prepare prepa-

rates of kallikrein by pancreas, Submaxillaris or urine.

Various methods are known for the production of partially purified kallikrein, e.g. according to German patent no. 890,857, kallikrein-containing glands can be thermolysed in aqueous suspension at temperatures between 38 and 65°C and the kallikrein-containing aqueous phases are separated from insoluble parts. For the production of partially purified kallikrein, according to German patent no. 910,580, urine and autolysates of pancreas or submaxillaris can also be combined with aqueous solutions of lead or zinc salts, the precipitate is separated, dissolved in phosphate buffer solutions, especially in diammonium hydrogen phosphate solutions, the solutions are dialysed, the dialysates are concentrated and the active substance is precipitated from the concentrates with organic solvents, e.g. with ethanol or acetone.

The still impure kallikrein preparations can e.g. according to German patent no. 1,102,973, it is further purified such that kallikrein is adsorbed at pH values from 3 to 10, especially at pH 6.0 to 7.5 on basic substituted celluloses, the basic substituted celluloses are separated and inactive by-products are washed out with a strongly diluted buffer solution. The active substance can be desorbed from the basic substituted celluloses with 1 to 5% electrolyte solutions. Preparations are available with approximately 50 to 100 KE/mg protein (KE = kallikrein unit). Determination of kallikrein unit by measuring blood pressure reduction in dogs (E. Frey, H. Kraut, E. Werle, Das Kallikrein-Kinin-System und seine Inhibitoren, F. Enke-Verlag Stuttgart 1968, page 11).

According to German patent no. 1,124,187, still very impure kallikrein preparations can also be subjected to electrodialysis using exchange membranes for further purification. Preparations with 50 KE/mg are thus produced.

Thus purified kallikrein preparations are only conditionally suitable for parenteral use. Firstly, the content of foreign proteins can lead to immunological sensitization and, after repeated use, to life-threatening anaphylactic shock. Secondly, with the previous purification methods it was difficult to obtain preparations that were sufficiently free of pyrogenic substances. It was also not possible to remove pyrogens by filtration using clear and sterile filter layers, as kallikrein is adsorbed by these materials, even

to a high degree.

Furthermore, it is already known that moons can produce smaller amounts of highly purified kallikrein using different methods. According to H. Fritz et al. (Hoppe Seyler's Z. Physiol. Chem. 348, 1120-1132 (1967)) a preparation with a specific activity of 1516 KE/mg protein is obtained by chromatographing a product purified according to German patent no. 910,580 one to three times on hydroxy1-apatite-cellulose. It is therefore necessary, however, to determine the optimal concentration of the elution buffer for each kallikrein sample and each new charge of the hydroxylapatite cellulose in preliminary experiments again. This method is also therefore technically not feasible. According to another proposal (H. Moriya et al., Biochem. Pharmacol. 18, 549-552

(1968). -precipitation with ammonium sulfate solution, precipitation with acetone, chromatography on hydroxylapatite and chromatography on "Sephadex"

G 100. This method is also technically unusable due to the small yields and the large number of steps.

The kallikrein produced according to this procedure on a laboratory scale is a mixture of two isoenzymes. By electrophoresis or by chromatography on anion exchangers, kallikrein can be separated into two components, kallikrein component A and kallikrein component B (E. Habermann, Hoppe-Seyler's Z. Physiol. Chemie 328, 15 - 23, F. Fiedler and E. Werle, ibid. 348 , 1097-1089 (1967)). It is not yet known whether the small structural difference lies in the amino acid sequence or in the carbohydrate part of the glycoprotein kallikrein. The two components cannot be separated in their biological effects.

Crystalline kallikrein as well as crystalline kallikrein components A and B have not yet been produced. The invention now relates to a simple method for the production of very pure, preferably crystalline kallikrein and possibly dividing the kallikrein into the pure, preferably crystalline kallikrein components A and B.

The invention therefore relates to one more method. production of highly pure, preferably crystalline kallikrein, optionally divided into its components A and B, whereby a kallikrein partially purified from pancreatic material by lead or zinc salt precipitation in a known manner is eluted with phosphate buffer salt solution and the eluate is desalted and chromatographed to cross-linked dextran substituted with diethylammonium methyl groups, or to cellulose substituted with diethylaminoethyl groups, in that the method is characterized in that the chronatogram is eluted at a pH value from 4.5 to 5 with ammonium acetate, ammonium formate, ammonium propionate or the corresponding alkylammonium salts and the eluate is then treated with cross-linked dextran substituted with carboxymethyl or sulfoethyl groups, that the thus purified solution of high-purity kallikrein is optionally divided in a manner known per se into components A and B and that the kallikrein is then purified from the thus prepared solution by adding a suitable precipitating agent, especially ammonium sulfate.

The determination of kallikrein's enzymatic activity takes place by measuring the hydrolysis of N-benzoyl-l-arginine ethyl ester (E. Werle and B. Kaufmann-Bretsch, Naturwissenschaften 46, 559 (1959)) in that of P.I.P. (Federation Internationale Pharmaceutique) standardized form, as a titrimetric test (Publication 1972, in J. mond. Pharm.)- For conversion of the enzymatic units in those when measuring blood pressure

ning in dogs certain biological kallikrein units (KE)

(E. Frey, H. Kraut, E. Werle, Das Kallikrein-Kinin-System und seine Inhibitoren, F. Enke, Stuttgart 1968, page 11) the factor 1 KE = 6.37 F.1.P. units is used. The protein content of solutions of the pure kallikrein is determined from the extinction at 280 nm, the extinction coefficient of E2gQ = 19.3 (= extinction of a 1% solution of kallikrein at pH 7.0) being used as a basis.

It was found that the desalination of the acc

German patent no. 910,580 produced eluate can take place by dialysis or by gel filtration. For the subsequent chromatography on macroporous anion exchangers, base-substituted celluloses or base-substituted gels are used according to the invention. sufficient pore width. Preferably, these purification steps are carried out on DEAE-"Sephadex" columns. The adsorption takes place at a pH of 4.0 to 8.0,

preferably at pH 4.5 to 5.0. The elution takes place by increasing the salt concentration at constant pH or lowering the pH value at constant salt concentration or a combination of both methods. , The change in pH and salt concentration can take place continuously as a gradient elution or stepwise. All known buffer salts can be used; volatile buffer salts are preferably used, e.g. ammonium formate, ammonium acetate, ammonium propionate or the corresponding alkylammonium salts, e.g. triethylammonium formate. IN

in a preferred embodiment of the method according to the invention, kallikrein is adsorbed from 0.2 M ammonium acetate pH 5.0 on DEAE-"Sephadex" in the form of a column, inactive protein is washed out with 0.25 M ammonium acetate pH 5.0 and then enriched kallikrein is eluted with 0.3 M ammonium acetate pH 4.5.

The chromatography on macroporous anion exchangers is a significant improvement of the method according to German patent no. 1,102,973, which is decisively surpassed in yield and simplicity when carried out on a technical scale.

The thus enriched kallikrein is further purified according to the invention by treatment with macroporous cation exchangers. Preparations of approx. 90% purity. As macroporous cation exchangers, acid substituted celluloses or acid substituted gels can in particular be used, e.g. of cross-linked dextrans or polyacrylamide, with sufficient pore width. Treatment of the solution of the enriched kallikrein with the cation exchanger can take place by stirring the solution with the exchanger and by filtering the solution through layers of the exchanger. If columns are used, it is appropriate to concentrate the solution of enriched kallikrein from the previous steps and to desalt. During stirring or during the filtration process, the gels retain by-products while kallikrein passes through the filter layer unimpeded.

The macroporous cation exchangers used for filtration or stirring are pretreated in a known manner. For filtration, they are stirred with diluted buffer solution and e.g. incorporated into columns. The column length/column diameter ratio is of secondary importance for the filtration process. Columns with a large column diameter are preferably used. For the preparation of the buffer solutions, the known buffer salts are used, especially however volatile buffer salts, e.g. ammonium formate, ammonium acetate, ammonium propionate or the corresponding alkylammonium salts, e.g. triethylammonium formate. The pH value of the buffer solutions can be chosen between 4.5 and 8.5, in particular between 5.0 and 7.0.

For the production of crystalline kallikrein, the highly purified kallikrein is crystallized by adding ammonium sulphate in solid form or as a concentrated solution with a pH value from 4.0 to 8.0. Crystallization starts at an ammonium sulphate saturation of approx. 45% within a few days, preferably at a temperature of 15 to 25<6>C. The crystals have the form of thin needles that cluster together in bouquets. The crystals can be separated from the mother liquor by centrifugation or filtration, washed with 50% saturated ammonium sulphate solution and dissolved in dilute salt solution or in water.

■The crystals consist of pure kallikrein. The specific activity of approx. 1600 KE/mg protein cannot be controlled further by several times of recrystallization.

For the production of the possibly crystalline kallikrein components, the purified kallikrein is divided according to the known methods by chromatography on DEAE-cellulose or DEAE-"SEPHA-DEX" into components A and B. You can also carry out separation of the components according to this method already at an earlier stage of the cleaning route and then separately further clean the separated components.

According to the invention, the highly purified kallikrein components are separated by adding ammonium sulfate in solid form or as a concentrated solution at a pH value of 4.0 - 8.0. Crystallization starts at an ammonium sulfate saturation of approx. 45% within a few days, preferably at a temperature of 15 - 25°C. The crystals have the shape of thin needles, which cluster together in bouquets. The crystals can be separated from the mother liquor by centrifugation or filtration, washing with 50% saturated ammonium sulphate solution and dissolved in dilute salt solution or in water.

The crystals consist of pure electrophoretically uniform kallikrein A resp. kallikrein B. Figure 1 shows the result of electrophoresis experiments. The work was done with veronal-Na buffer (pH 8.6). Ionic strength was 0.075, voltage: 100 volts, duration: 20 minutes. At time 0' it was applied at the height of the dashed line. At 1, kallikrein was used, at 2, kallikrein component A and at 3, kallikrein component B was used. The specific activity of approx. 1600 KE/mg protein cannot be increased further, not even by repeated recrystallization.

Surprisingly, this method according to the invention succeeds in separating partially purified kallikrein from its by-products and from foreign enzymes very simply and without the use of cumbersome elution methods. It could not be foreseen that the by-products and foreign enzymes, but not the kallikrein, are retained by the macroporous cation exchangers and the kallikrein remaining in solution is obtained by the method according to the invention in very good yields at high purity. During crystallization of the two kallikrein components A and B, pyrogenic substances that are difficult to remove are also almost completely separated. The simplicity of the method according to the invention enables implementation also on a technical scale. The method according to the invention is thus an enrichment of the technique.

The dissolution of the kallikrein and kallikrein component crystals is characterized by a UV spectrum at pH 7 with a maximum at 282 mm, a minimum at 251 mm, 2 shoulders at 277 and 290 mm and a ratio of the extinctions of 280 and 260 mm of 1 ,78.

(See figure 2, not broken line).

In 0.1 n NaOH, the UV spectrum shows two maxima at 280 and

289 mm (see Figure 2, dashed curve).

The crystalline kallikrein according to the invention as well as

the crystalline kallikrein components A and B can also be used as active ingredients of pharmaceuticals. Due to their karut-widening effect, kallikrein-containing drugs are used as already known, especially in peripheral blood circulation disorders, e.g. Endangiitis obliterans, Arteriosclerosis obliterans, Morbus Raynaud, in disturbed fracture and wound healing, e.g. Sudecksche's Syndrome, burns and circulatory disorders due to age

with results.

The application can take place in the form of tablets or dragees containing the crystalline kallikrein or its components in admixture with inert, non-toxic, pharmaceutical suitable carriers or parenterally as intramuscular or intravenous injections of an aqueous solution of crystalline kallikrein or its components. Especially for parenteral use, crystalline kallikrein offers the advantage of much less risk of unexpected side effects and allergic reactions compared to less pure preparations.

By adding high molecular weight kiloiden according to German patent no. 941,685, e.g. polyvinylpyrrolidone or dextran or by forming complexes with the kallikrein inhibitor "Trasylol" according to German patent no. 1,000,566, crystalline kallikrein can be used to produce delayed-acting injection preparations. Kallikrein for injection purposes can be prepared as well as ready-to-use isotonic solutions and also in solid form by freeze-drying a mixture of crystalline kallikrein or crystalline kallikrein components with suitable carriers, e.g. dextran, mannitol, lactose, gelatin, of which, by dissolving in a suitable solvent, the ready-to-use solution is prepared immediately before use. Examples • : a) 200 liters of a pig pancreas produced and dialyzed eluate of a lead salt precipitate, containing 10.8 Mio. KE, was evaporated in a thin layer evaporator to 15 liters and then desalted by gel filtration over a column of "Sephadex" G -25- The desalted solution was adjusted to pH 5.0 with acetic acid and a small precipitate formed thereby filtered off. Solid ammonium acetate was then added to a specific conductivity of 12 mS. This solution was passed over a 20 x 65 cm

column (20 liters) of DEAE-"Sephadex" A-50, equilibrated with 0.2 M ammonium acetate pH 5.0, as kallikrein is adsorbed. The column was then first eluted with approx. 40 liters o.25 M ammonium acetate pH 5.0 and then with 0.3 M ammonium acetate pH 4.5. Kallikrein appears in the eluate shortly after the pH has dropped to approx. 4.5, (figure 3)-The active fractions contained 9.35 million KE (86.5%). The solution was concentrated in vacuo and desalted over a column of "Sephadex" G-25, a lyophilized sample having a specific activity of 414 KE/mg weight.

Explanations for Figure 3.

Ordinate units:

0 - 800 : Kallikrein units (KE) per ml solution,

100 -.20 : UV transmission in percent (wavelength 280 mm),

4.2 - 5.0 : pH value

Abscissa unit: fraction number.

The continuous curve between fractions 5 and 45 shows the course of the ultraviolet absorption during the elution process, the dashed curve corresponds to the course of the pH value. The connecting line of the triangle corresponds to the course of activity during the elution, indication 1 KE/ml.

b) The desalted solution with a volume of 300 ml was filtered over a 10 x 130 cm column (10 liters) of CM-"Sephadex" C-50, equilibrated with 0.01 M ammonium acetate pH 5.0. In the eluate, kallikrein appeared first, full of inactive contaminants (figure 4). The kallikrein fraction (930 ml) contained 5.5 million KE (59%). A lyophilized sample had the specific activity of 1060 KE/mg weighing resp. 1220 KE/mg protein. According to DAB, the febrile effect amounted to 1.85 (Z(A T)<2> in 3 animals).

Figure 4.

Ordinate units:

0 - 800: KE per ml solution,

100 - 20: UV transmission in percent (wavelength 280 mm), Abscissa unit: fraction number.

The connecting line of the triangle corresponds to the course of activity of the eluate, indicated in KE/ml. The second curve shows the course of the UV absorption.

c) Preparation of crystalline kallikrein:

A portion of the solution, containing 115,000 KE, was concentrated to 5 ml and mixed with saturated ammonium sulfate to 45% saturation. After 6 days at 20 to 25°C, crystallization started. After 3 weeks, the crystals were centrifuged, washed once with 5 ml of 50% saturated ammonium sulfate solution and dissolved in 0.05 molar phosphate buffer pH 7.0. The specific activity of kallikrein in this solution was 1560 KE/mg protein. The yield was 71%, the fever effect according to DAB 0.32.

d) Preparation of crystalline kallikrein component B:

4.42 million KE of a solution of highly purified kallikrein obtained according to point b), was adsorbed on a column 5 x 100 cm of DEAE-"Sephadex" A-50', equilibrated with 0.2 molar ammonium acetate, pH 6.7. The elution took place with a convex gradient 0.2 to 0.55 molar ammonium acetate pH 6.7, prepared with a 2 liter mixing vessel. In the eluate, two slightly overlapping activity peaks of kallikrein appeared, the first of which was identified electrophoretically as kallikrein component B, the second as kallikrein component A. The pure kallikrein A resp. B-containing fractions were combined. There were formed: 2.1 million KE kallikrein component B (47.5%), 1.25 million KE kallikrein component A (28%) and 0.32 million KE" of a mixture of kallikrein component A and B of the overlap area of the peak (7.2%), a total of 83%.

A portion of the solution containing kallikrein component B was evaporated to a concentration of approx. 20,000 KE/ml and mixed with saturated ammonium sulphate solution to 45% saturation. After 6 days at 20 to 25°C, crystallization starts. After 4 weeks, the crystals were centrifuged off, washed with 50% saturated ammonium sulfate solution and dissolved in 0.05 molar phosphate buffer pH 7j0. The specific activity was 1650 KE/mg protein, yield 64%.

e) Preparation of the crystalline kallikrein component A:

In the same way as described under point d), was

also kallikrein A crystallized. There is no difference in the microscopic appearance of the crystals of kallikrein A and kallikrein B.

Claims (1)

Process for the production of highly pure, preferably crystalline kallikrein, possibly divided into its components A and B, whereby a kallikrein partially purified from pancreatic material in a known manner by lead or zinc salt precipitation is eluted with phosphate buffer salt solution and the eluate is desalted and chromatographed on cross-linked dextran substituted with diethylammonium methyl groups, or on cellulose substituted with diethylaminoethyl groups, characterized in that the chromatogram is eluted at a pH value from 4.5 to 5 with ammonium acetate, ammonium formate, ammonium propionate or the corresponding alkylammonium salts and that the eluate is then treated with cross-linked dextran substituted with carboxymethyl or sulfoethyl groups, that the thus purified solution of highly pure kallikrein is optionally split in a manner known per se into components A and B and that the kallikrein is then optionally recrystallized in the form of its split components A and B from the thus prepared solution by adding a suitable a precipitating agent, especially ammonium sulfate.