HU183432B - Process for the specific elution of polypeptides with callicreine-inhiaitor and trypsine-inhibitor activity from adsorbents of silicate type - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a specific method for the specific release of kallikrein inhibitor and trypsin inhibitory polypeptides from silicate-type adsorbents, an organic polar solvent and an acidified mixture of water at pH 1 to 3, characterized in that it is an aqueous solvent mixture acidified to pH 1-3 as a release solvent. which comprises, in addition to water, 5-95% Lewisbase not proton-containing in an acidic aqueous medium, and optionally a protic organic solvent, preferably methanol, ethanol or acetone, instead of a portion of this Lewis base. With the new method, the active ingredient can be obtained with better efficacy and greater purity than previously known methods. -1-

Description

The present invention relates to a novel process for the specific release of kallikrein inhibitor and trypsin inhibitor polypeptides from a silicate-type adsorbent.

Several methods are known for the recovery and purification of kallikrein inhibitor and trypsin inhibitory polypeptides in animal carbon extracts by binding to, or dissolving from, adsorbent, ion exchanger, or immobilized enzyme.

In the process of the Hungarian Patent No. 153,247, the active ingredient is bound from the organic extract to a silicate-type adsorbent from a pH of 5 to 7 and then from the adsorbent to a pH of 30-90% diluted with a polar organic solvent diluted with water. Carbon alcohol or ketone.

The disadvantage of the process is that while the adsorbent binds almost quantitatively to the active polypeptide, it also adsorbs a large amount of ballast (cf. Austrian Patent No. 307,615, page 3, lines 56-58) and the solvent is poor effective (50-60%) and non-specific dissolution of the active ingredient, so the degree of purification is not satisfactory.

German Patent Publication No. 2,509,482 discloses a process in which trypsin inhibitory polypeptides are bound by immobilized trypsin and resolved by varying the pH. The disadvantage of the process is that a very large amount of immobilized enzyme is required for binding, which makes it questionable on a large scale. Another disadvantage is that during capture and release, the inhibitory polypeptide produces a partially inactive artificial product which reduces yield and complicates further purification steps. Thus, this method is not suitable for purifying crude organ extracts.

According to GD 76,773, the protease inhibitor polypeptide is bound from the organic extract to activated carbon and lysed with 1 to 20% lower aliphatic monocarboxylic acid or mineral acid at 20 to 60 ° C. in the presence of% phenol. The disadvantage of the process is that it allows only a very large volume of the active ingredient to be dissolved, so that it is hardly concentrated, and further recovery of the active ingredient requires 2 to 2.5 times the volume of the organic extract in acetone.

According to the method disclosed in German Patent Application No. 2,201,088, the kallikrein-trypsin inhibitor polypeptide is bound from its solution on a polystyrene-based amphoteric ion exchanger, and the active ingredient is released by chromatography on a salt gradient. The disadvantage of the process is that the binding on the ion exchanger is greatly influenced by the inorganic and organic ions present in the solution. In the example described herein, binding is performed using a solution of 2000 KIE / mg protein (CIU: kallikrein inhibitor unit, TIE: trypsin inhibitor unit) which has been purified in advance and not crude. Another drawback is that salt gradient chromatography is difficult to achieve on a large scale.

SUMMARY OF THE INVENTION The object of the present invention was to provide a method which overcomes the drawbacks of the known methods and allows the recovery of the polypeptide having kallikrein and trypsin inhibitory activity from animal organ extracts in good yield and purity by a method which is readily commercially available.

This object is achieved by the use of silicone adsorbent-bound kallikrein and trypsin inhibitory polypeptides 2 from an animal organ extract, a Lewis base and water, or optionally a Lewis base, which is more difficult to protonate than water in an aqueous medium, dissolve in the adsorbent using a mixture of a protic solvent and water to pH 1-3. By Lewis base we mean compounds which are capable of binding protons or coordinate bonds through their release electron pair (cf. Szántay, Theoretical Organic Chemistry, Budapest 1975, p. 161). More difficult than water is the protonization of Lewis bases which do not take up a proton in acidic aqueous media and thus retain their ability to form a coordinate bond. (Lewis bases which are protonated in acidic aqueous media do not facilitate dissolution of the peptides from the adsorbent in the process of the invention.) As the Lewis base more difficult to protonate than water, the process of the invention preferably has N-substituted or unsubstituted carboxylic acid amides, carboxylic cyclic ethers or thioethers. Preferably, the protic solvent is ethanol or methanol. The amount of Lewis base in the dissolving aqueous solvent mixture may be from 5% to 95%, and if desired, the protic organic solvent may be used in place of a portion of the Lewis base (based on the weight of the total solvent mixture); hydrochloric acid is conveniently used to adjust the pH.

The process of the present invention is applicable to any known method of recovering kallikrein and trypsin inhibitors, wherein the basic extracts of kallikrein and trypsin inhibitors from an appropriate animal organ such as bovine lung, pancreas, parotid or liver are of a silicate type. adsorbent, such as bentonitone, montmorillonitone or the like, and then selectively dissolve the adsorbent therefrom. Such procedures are described, for example, in Hungarian Patent Nos. 153,140, German Patent 2,447,050 and Swiss Patent No. 519,914.

Thus, according to the present invention, an aqueous solvent mixture which is acidified to a pH of 1 to 3 is used to selectively dissolve the desired active ingredient from the adsorbent, with 5 to 95% water in a non-proton Lewis base and optionally part of this Lewis base. containing a protic organic solvent, preferably methanol, ethanol or acetone.

Preferably, the polypeptide to be recovered as the active ingredient is dissolved in a silicate-type adsorbent in an aqueous solution containing from 20% to 90% Lewis base, acidified to pH 1-3. The polypeptide to be recovered as the active ingredient may also be dissolved in an aqueous solution of at least 5% Lewis base and 20-70% ethanol or methanol acidified to pH 1-3.

In particular, the Lewis base is preferably dimethylformamide, dimethylsulfoxide, tetrahydrofuran, mercaptoethanol, acetamide, acetonitrile or dioxane.

The process according to the invention has the advantage, in particular, that the polypeptide active ingredient can be obtained with a much better efficiency and purity than the previous processes. According to the solubilization process disclosed in Hungarian Patent No. 153,247, between 54% and 60% of the polypeptide surfactant in the extract can be liberated from the silicate-based adsorbent. In contrast, by using Lewis bases in the solvent according to the present process, the yield can be increased to 80-95% and the product obtained is essentially

183,432 sen cleaner. Starting from lung extract of the same purity, the procedure according to Hungarian Patent No. 153,247 yielded an average of 2.3 TIE / g and 0.45-0.8 million KIE / g protein purity product, respectively. , 1 million KIE / g or 4.2-8.0 ΤΓΕ / g protein product (TIE: trypsin inhibitor unit; KIE: kallikrein inhibitor unit).

A further advantage of the process according to the invention is that it is also possible to use substances which, in the case of acetone precipitation used to recover the polypeptides, allow the recovery of the acetone used in the precipitation after separation of the polypeptide precipitate. The difference between the boiling points of ethanol (fp: 78.3 ° C) used in the previous process and the acetone (fp: 56.3 ° C) used for the precipitation is 22 ° C, which makes the regenerability of the solvents so difficult that it is no longer soluble and economically. In contrast, the use of higher boiling Lewis bases for solubilization avoids the use of low-alcohol alcohols close to acetone, so that the acetone used for precipitation and the Lewis base used for leaching can be easily separated by distillation. Suitable Lewis bases include, for example, dimethylformamide boiling point 153 ° C, dioxane 106 ° C, acetamide 221 ° C. The recoverability of the solvents thus afforded a very significant economic advantage.

Practical embodiments of the process of the invention are illustrated by the following examples. The following methods were used to determine the efficacy and purity of the products prepared by the examples:

The kallikrein inhibitor activity (CI) was according to Werle-Kaufmann-Boetsch [Hoppe-Seyler's Z.f.physiol. Chem. 319, 52 (1960)] and trypsin inhibitor activity (TIE) according to Anson [J. Gene. Physiol. 22, 79 (1949)]. Protein content was calculated from extinction at 280 gm.

Example 1

An aqueous extract of 100 kg of bovine milk with 400 liters of aqueous extract containing a total of 150 million KIU and 577 TIE activity polypeptides is adjusted to pH 5.6 with calcium hydroxide, and then 4 kg of acid-activated bentonite are added to the solution and stirred for one hour. The adsorbent was then filtered off and washed with water, and the mixture was stirred with 10 liters of aqueous ethanol, 10% v / v dimethylformamide and 0.2 N HCl (pH 0.8) for half an hour, filtered and filtered. repeat the operation with 5 liters of the same aqueous mixture. The filtrates were combined and the polypeptide drug was precipitated with three volumes of acetone and the precipitate was collected by filtration and air dried. The acetone precipitated product had a total activity of 138 million CIs and 530 TIEs, with a purity of protein of 1.1 million KIE / g and 4.23 TIE / g, respectively, corresponding to a 92% yield. From this dry precipitate, the inhibitory polypeptide can be recovered by known methods. for example, by mixing with 90% ethanol at 10 times the dry precipitate weight, filtering and repeating the process with 5 times the precipitate weight with 90% ethanol, and then adding to the combined filtrate two and a half volumes of acetone to precipitate the polypeptide.

Example 2

From the aqueous extract of 100 kg of bovine lung, the crude active ingredient was adsorbed to 4 kg of acid-activated bentonite as described in Example 1. The dissolution was performed with 10 L followed by 5 L of 0.1 N aqueous hydrochloric acid (pH 1.45) containing 70% methanol and 10% acetamide, the two leaching liquids were combined and the polypeptide active compound was precipitated by adding three volumes of acetone. . The resulting precipitate was filtered off, dried and dissolved. The product thus obtained contains 138 million CIs and 530 TIE activities, corresponding to 92% of the active ingredient content of the starting extract. The product has a purity of 1.6 million KIE / g or 6.1 TIE / g protein.

Example 3 From a 40 liter aqueous extract from a kg of bovine lung containing 20.5 million KIU and 78.8 TIE activity polypeptide, the active ingredient polypeptide was adsorbed on 600 g of ground montmorillon. The adsorbent was washed with water and eluted with a 10-fold mixture of 90% dimethylformamide and 10% 0.5 N aqueous hydrochloric acid (pH 2.1). The eluate was precipitated from the eluate by the addition of two and a half volumes of acetone, the precipitate was collected, dried and purified by dissolving it in 90% alcohol and re-precipitating with acetone as described in Example I. The product thus obtained has a purity of 17.4 million IU and 67 TIE and a purity of 2.4 million IU / g protein or 9.2 TIE / g protein. Yield of crude lung extract activity: 85%.

EXAMPLE 4 5 L of aqueous extract of Bovine Parotid, containing 1.9 million CIs and 7.26 TIE activity, was adjusted to pH 5.6 with calcium hydroxide and from this aqueous extract the active substance was treated with acid-treated bentonite. adsorbing. The adsorbent-bound active ingredient was eluted with a mixture of 40% v / v ethanol, 30% v / v dimethylsulfoxide and 30% v / v water and acidified to pH 2.5 with hydrochloric acid. From the eluate, the product precipitated with acetone according to the previous examples was 1.68 million KJE and 6.42 TIE active. The active ingredient polypeptide has a purity of 1.85 million KIE / g protein or 7.1 TIE / g protein. Yield of crude parotis extract: 88%.

EXAMPLE 5 A polypeptide was adsorbed on 400 g of ground acid-treated bentonite from an aqueous extract of kg bovine pancreas containing 8.5 million CIs and 32.7 TIE activity. Dissolution is carried out with 4 liters of a mixture of 60% dioxane and 40% aqueous hydrochloric acid, the pH of the mixture is adjusted to 2.5 and the pH is repeated several times with stirring for one hour. adjust by addition of 2N hydrochloric acid. The eluate was precipitated with three volumes of acetone, and the precipitate was collected and dried. The product contains total activity of 6.8 million CIUs and 26.1 TIEs, with a purity of 2.1 million CIU / g and 8.2 TIE / g protein; Yield for crude pancreas extract: 80%.

Example 6: 50 liters of aqueous extract from kg bovine lung containing 17 million CIF and 65 TIE activity polypeptides

183,432 contains 400 g of acid-treated bentonite to adsorb the inhibitor polypeptide. After adsorption, the btontonite was collected and stirred with 4 liters of 90% dimethylformamide and 10% water for 1 hour while maintaining the pH of the mixture at 3.0 with 2N hydrochloric acid. The bentonite is then isolated by filtration. The filtrate was treated with two and a half volumes of acetone, and the precipitate was filtered off, washed with 400 ml of acetone and dried. The active compound polypeptide was then dissolved in 90% ethanol as in Example 1 and precipitated from the solution with acetone. The product contains 14 million ICUs and 54 TIE activities. 2.6 million KIE / g and 10 TIE / g protein purity. Yield calculated for activity of crude organ extract; 82%.

Claims (4)

Patent claims CLAIMS 1. A process for the specific dissolution of a kallikrein inhibitor and a trypsin inhibitor polypeptide from a silicate-type adsorbent in an acidic mixture of an organic polar solvent and water to a pH of 1-3, characterized in that the aqueous solvent mixture is a pH of 1-3. containing 55 to 95% of a non-proton Lewis base in an acidic aqueous medium and optionally replacing part of this Lewis base with a protic organic solvent, preferably methanol, ethanol or acetone. 10 2. The process of claim 1, wherein the dissolving solvent mixture comprises 50-90% Lewis base and 10-50% water. 3. The process of claim 1 wherein the dissolving solvent mixture is water and 15 containing at least 5% Lewis base, 20-70% methanol or ethanol. Process according to any one of the preceding claims, characterized in that the Lewis base is dimethylformamide, acetamide, dimethylsulfoxide or dioxane.