IL46587A - Process for the isolation of a polyvalent proteinase inhibitor - Google Patents

Description

PROCESS FOR THE ISOLATION OF A POLYVALENT PROTEINASE INHIBITOR (HOE 74/B 002) The present invention relates to the isolation of a polyvalent inhibitor of proteinases from animal organs.

The basic polyvalent proteinase inhibitor isolated from ani^ mal organs has an outstanding importance in therapy owing to its property of inhibiting trypsin, chymotrypsin, the kallikreins of plasma, organs and urine, as well as plasmin.

Since the discovery of this inhibitor, a great number of processes for isolating it have become known. After extraction of the organs, the inhibitor is isolated from the extract by fractionation with organic solvents such as alcohol or acetone, with the aid of various protein precipitating agents such as sul-foealicylic acid, thiosalicylic acid, trichloroacetic acid, meta-phosphoric acid or various salts, by specific adsorption or by chromatographic methods. However, most of these processes are unsuitable for an operation on an industrial scale, if it is required to obtain goad yields and to have to p«srform the process in simple way at the same time.

A process which essentially satisfies these two requirements comprises the adsorption of the proteinase inhibitor of the organ extract on carboxymethyl-cellulose. However, the process step of adsorption on carboxymethyl-cellulose requires a low ion strength of the solution which can be attained only by strong dilution of the organ extract. In addition to this disadvantage of having to operate with large volumes and the great amount of ion exchanger, the filtrability of the strongly swelling carboxymethyl-cellulose represents technically a difficult problem which is the more serious in the regeneration which must be carried out in the alkaline pH-range. In view of the known basic properties of the polyvalent proteinase inhibitor, it seemed self-suggesting to use for the isolation of this substance, instead of carboxy-methyl-cellulose, other acid ion exchangers which have the form^ of a resin and which do not have the above-described disadvantage. The methods described in the literature in this respect, however, proved very unsatisfactory. For the adsorption of the inhibitor, a large amount of exchanger resin is required, while the yields are relatively poor and the product obtained has no satisfactory purity, or a very pure product is prepared with considerably expensive fractionation measures without regard to the yield, which would be inapplicable on an industrial scale.

Now, we have found that cation exchangers which carry sulfonic acid groups or phosphonic acid groups as functional groups are particularly suited for the isolation of the polyvalent proteinase inhibitor.

A copolymer gel of ethene-sulfonate and acrylamide may be used with advantage as adsorbant. This gel, which can be prepared according to known processes as described in Example 1 of the present specification, is a copolymer of ethene-sulfonic acid and acrylamide (in a weight proportion of about 20:1 to 2:1, preferably 8:1 to 3:1) which is cross-linked with 1 to 15 % of formaldehyde. The grain size suitable for the adsorption of the proteinase inhibitor is O.06 to 3 mm.

Well suitable for the process of the invention have also proved the commercial polyphenol- (ΛΓ -sulfonic acid ion exchangers, furthermore polystyrene-sulfonic acid resins, in particular those whose capacity is increased owing to a macro—porous structure, or polystyrene-phosphon c acid resins. A grain size of these ion exchangers of about 0.03 to 0.08 mm has shown to be particularly advantageous.

The exchanger types of the present invention are distinguished over carboxymethyl-cellulose by a considerably higher adsorptive capacity. ^ Furthermore, the filtration speed as compared to that with carboxymethyl-cellulose for the separation of not-adsorbed material as well as for the elution of the inhibitor from the resins used according to the invention can be essentially augmented. The ion exchangers mentioned can be easily regenerated after elution of the inhibitor, since the through-flow speed of solutions is also not impeded in the alkaline range. The exchangers may be used several times for the isolation of the polyvalent proteinase inhibitor. This can be effected according to the batch method or according to the column method.

Accordingly, the object of the present invention is a pro- s cess for the isolation of a polyvalent proteinase inhibitor from its aqueous solutions, preferably from an aqueous extract of animal organs, preferably from bovine lungs, which comprises adsorbing the inhibitor on an ion exchanger resin with functional sulfonic acid groups or phosphonic acid groups, at pH-values of from 0.5 to 10.5, preferably pH-values of from 7.5 to 9.5, separating the adsorbant and subsequently eluting it with aqueous saline solutions at pH-values of 1 to 13, preferably pH-values of from 10 to 12.5; in proximity of pH 12.5, the addition of salt may be omitted, whereas with falling pH-value increasing amounts of salt are to be added in order to augment the elutive capacity. If the ethene-sulfonate/acrylamide copolymer gel is used, there are added, for example at a pH-value of 3, about 10% of sodium chloride to the eluting medium. If polyphenol- ^-sulfonic acid ion exchangers or polystyrene-phosphonic acid resins or polystyrene-sulfonic acid acid resins are used, the elution may be carried out, for example at pH 7.0, with addition of about 20% of sodium chloride or such-amounts of another salt which provide a comparable elution ca- ^ pacity. With these types of exchangers, it must be taken into consideration that, owing to the higher salt concentrations required for the elution, precipitations of the inhibitor may occur at elutions at pH-values below 5, which must be compensated by an increased elution volume in order to keep losses of yield as low as possible.

The process of the invention is used in the isolation of the proteinase inhibitor; it is of advantage to use as starting material for the adsorption of the proteinase inhibitor a pre-purified extract of animal organs.

For determining the proteinase inhibitor isolated according to the invention, the inhibition of plasmin with casein as the substrate may be used. This test determines how much the enzy-matical activity of a plasmin, previously determined according to the method described by L.F. Remmert and P.P- Cohen, J. Biol. Chem. 181, 431 (1949), is reduced by a quantity of inhibitor added. The inhibition of a plasmin unit is defined as anti-plasmin unit. With a view to the specific activity of the proteinase inhibitor, the anti-plasmin units (APU)/ml are referred to an extinction value of the solution of the inhibitor of 1.0 measured at a wave length of 280 nm. Consequently, referred to APU/mg of nitrogen, a purification factor of 20 to 35 is attained according to the process of the invention.

The eluate obtained according to the invention may be further purified by gel chromatography, whereupon the inhibitor is obtained in high purity and with good yield. Thus, an inhibitor of about 170 APU/ml at E2so β 1,0 can De obtained« This purity of the polyvalent proteinase inhibitor permits its therapeutic use in humans. t The following Examples illustrate the invention.

EXAMPLE 1: A. Preparation of a copolymer of ethene-sulfonate and acryl amide : 85 parts by weight of 25% aqueous solution of the Na-salt of ethene-sulfonic acid were adjusted to pH 6.9 with 40% H2S04. Then, 5.3 parts by weight of acryl-amide were dissolved in this solution and the whole was heated to 45° C. Polymerization was initiated by the addition of 0.425 part by weight of ammonium-peroxyde-disulfate and 0.085 part by weight of sodium disulfite. The batch heated up to 98° C. In order to complete the polymerization, 0.0425 part by weight of ammonium-peroxyde-disulfate and 0.0085 part by weight of sodium disulfite were added, the whole was stirred for 2 hours at 85° C and then cooled.

B. Cross-linking with formaldehyde and thermical after-treatment 13.6 parts by weight of a 28% formaldehyde solution were added to the solution of copolymer and allowed to stand for 15 hours at 25° C. The gel obtained was dried at 80° C under reduced pressure and the dry product was heated for 30 minutes to 150° C. It was then ground and sieved. The fraction having a grain size of 0,06 to 0.3 mm was found to be especially suitable for the isolation of the polyvalent proteinase inhibitor. Before use, the cross-linked product was swelled by washing with desalted water and freed from water- soluble by-products.

EXAMPLE 2: 33 kg of the wet ethene-sulfonate/acryl-amide copolymer ge^L (prepared as described in Example 1) were added to 1650 1 of a g crude extract of lung tissue which contained 12 x 10 antiplasmin units. The mixture was stirred for 30 minutes and then allowed to stand for 30 minutes to permit sedimentation of the adsorbant. Then, 1250 1 of the supernatant could be decanted off. In order to remove the residual supernatant, the adsorbant was filtered off. Elution of the proteinase inhibitor was effected with 120 1 of de- ionized water, the pH-value of the suspension having been adjusted to 12.5 by means of concentrated sodium hydroxide solution. The eluate contained 95% of the quantity of inhibitor present in the extract in a purity of 60 APU/ml at E280 "* 1,0.

The eluate obtained in the afore-described manner could be freed from higher molecular impurities advantageously by gel chromatography, for example on Sephadex^ G-50 (Deutsche Pharmacia, Frankfurt/M. ) . Thereby, tix@ active substance was obtained with a purity of 140-200 APU/ml at A ¾80 β 1,0 and in a yield of more than 50%, referred to the raw extract.

If desired or required, the proteinase inhibitor so obtained may be subjected to a process for removin pyrogens, adjusted to a therapeutically usual concentration of 250 APU/ml, filtered under sterile conditions and filled into ampuls.

In this form, the proteinase inhibitor may be administered parenterally , in particular intravenously.

EXAMPLES 3 to 11: 5 Liters each of a lung tissue raw extract containing about 6 APU/ml were combined with different amounts of various ion exchangers and worked up essentially as described in Example 2.

The following compilation shows, by way of example, some of the tests carried out. i Ion exchanger Reaction Proteinase conditions inhibitor Example Designation Functional Quantity Ads. Eluant 'yield Purity group g/1 at % APU/ml PH at E280= 1 3 Copolymer Sulfonic 20 8.5 Water/ 92 60 gel acc.to acid 10% NaCl Example 1 pH 3.0 4 Copolymer Sulfonic 20 8.5 Water 81 70 gel acc. to acid pH 12.5 Ex. 1 5 Copolymer Sulfonic 20 8.5 Water/ 82 71 gel acc. to acid 10% NaCl Ex. 1 pH 4.5 6 Copolymer Sulfonic 20 4.5 Water 92 10 gel acc.to acid pH 12.5 Ex. 1 7 Copolymer Sulfonic 20 4.5 Water/ 85 13 gel acc.to acid 10% NaCl Ex. 1 pH 4.5 8 Bio-Rex(R) Sulfonic 2 8.5 Water 90 53 40 *) acid pH 12.5 9 Bio-Rex (R Sulfonic 2 8.5 Water/ 65 52 40 *) acid 20% NaCl pH 7.0 10 AG-MP-50^* Sulfonic 4 8.5 Water 94 32 ac d pH 12.5 11 Bio-Re ^ Phosphonic 6 8.5 Water 75 53 63 *) acid pH 12.5 *) Bio-Rad Laboratories, Richmond, California, U.S.A.

Claims (11)

We claim:

1. ) A process for the isolation of a polyvalent proteinase inhibitor from animal organs, which comprises adsorbing the inhibitor from its aqueous solutions at pH 0.5 to 10.5 on an ion exchanger containing functional sulfonate or phosphonate groups and eluting it with water or a salt solution at pH 1 to pH 13.

2. ) A process as claimed in claim 1), wherein a copolymer gel of ethene-sulfonate and acryl-amide is used as adsorbant.

3. ) A process as claimed in claims 1 and 2, wherein a gel prepared by copolymerization of ethene-sulfonic acid and acryl-amide in a proportion by weight of 20:1 to 2:1 and subsequent cross-linking with 1 to 15% of formaldehyde is used for the adsorption.

4. ) A process as claimed in claim 1), wherein a polyphenol- Lj~ -sulfonic acid ion exchanger is used as adsorbant.

5. ) A process as claimed in claim 1), wherein a polystyrene/ sulfonic acid ion exchanger, preferably with macro-porous structure, is used as adsorbant.

6. ) A process as claimed in claim 1), wherein a polystyrene/ phosphonic acid ion exchanger is used as adsorbant.

7. ) A process as claimed in claims 1 to 6, wherein the elution is effected at about pH 12.5 with water without addition of salts.

8. ) A process as claimed in claims 1 to 6, wherein the eluting solutions contain increasing amounts of salt with falling pH-value. ^

9. ) A process as claimed in claim 8, wherein, if the copolymer gel of ethene-sulfonate/acryl-amide of claim 3) is used, the elution of the proteinase inhibitor is carried out with a 10% sodium chloride solution at pH 3.0.

10. ) A process as claimed in claim £&) , wherein, if polyphenol- -sulfonic acid ion exchangers, polystyrene-sulfonic acid resins or polystyrene-phosphonic acid resins are used, the elution of the proteinase inhibitor is carried out with a 20% sodium chloride solution at pH 7.0.

11. ) A polyvalent proteinase inhibitor, prepared according to any one of the processes claimed in claims 1 to 10). COHEN ZEDE & SPISBACH P.0. Box 331 1 6 , Tel- A v i v Attorneys for Applicant O ~