CA1053661A - Protease-free proteins, and methods of manufacturing and using the same - Google Patents

Abstract

Abstract Protease-free proteins, especially protease-free kallikrein, are prepared by contacting a solution of the protein with a carrier-bound water-insoluble protease inhibitor that does not bind to said protein. The protease-free proteins have improved storage stability in solution, and are used therapeutically for the same purposes as the protein before treatment according to the invention.

Description

1~53i>61 The present invention relates to protea~e-free protein, psrticularly kallikreln, golution~; a procesR for their prepara-tion; and their use as medicaments.
When kallikrein acts on endogenic kininogen, lt S liberates the physiologically active kinins (for example kallidine). Kallikrein preparations are therefore used thera-peu~ically [E.K. Frey, H. Kraut and E. Werle, "Das Xallikrein-Kinin-System and seine Inhibitoren" (The Xallikrein-Kinin System and Its Inhibitors), F. Enke, Stuttgart, 1968, page 150 et seq.l.
It ha~ already been disclo~ed that kalllkrein can be prepared, for example, from pancreas glands of pigs in accordance with the process of German Published Specification 2,154,557.
It i8 prefer~bly admini~tered orally or by intramuscular or intravenous inJection.
Hitherto ~t ha8, however, not been possible to prepare in~ection solutions which can be kept for prolonged periods.
The kallikrein solution filled into ampoules was therefore usually brought to a dry form, and hence to the more stable solid form, for example by freeze-drying, and the solid kallikrein was dissolved in a suitable isotonic solution before use.
This procedure i8 involved and time-consuming, and in addition has the disadvantage that the small, physiologic~lly active amount~ of kallikrein can only be freeze-dried in the presence of inert additives acting as structure-forming agents, for example polyvinylpyrrolidone, mannitol, dextran, lactose and others; thus when used therapeutically, undesired foreign substances are administered together with the active compound.

'10536~
It ha8 also been possible hltherto Ln principle to achieve stabilization of the solutions of kallLkrein by adding to the Icallikrein solution ~uit~ble inhibitors which do not inhibit kallikrein and thereby inactivate the protease impurities.
However, such additions of foreign substances exhibiting biological activity are most undesirable in a medicament.
It is therefore an ob~ect of th0 invention to provide a process for producing very pure solutions of kallikrein, free from foreign additives, which are stable for prolonged periods.
This is accomplished by the present invention by the provision of protease-free kallikrein. Desirably, the protease content should be as low as possible, with good results being obtained at a protease content of not more than 0.011 U/mg, e.g. from 0.004 to 0.011 U/mg, for kallikrein having an activity of1000-1300 KU/mg.
The present invention is ba~ed on the discovery that even the purest preparations of kallikrein which have hitherto been disclosed still contain traces of proteolytic enzymes in an order of magnitude of <1% and that, surpri~ingly, there is a direct relationship between the protease content of various kallikrein preparations and their stability in solution. The loss in kallikrein activity on storing the solution i8, as we h~ve found, attributable to a destruction of the kallikrein molecule by proteases.
~lthough protease impurities can be removed by the relatively difficult conventional purification processes, for example ion exch~nge chromatography, the conventional processes

-2-1~5 3~ ~ 1 are often accompanied by great losses 9~ kallikrein activity.
Surprisingly it Ls possible, in aecordance with a further feat~re of the invent~on, to stabilize ~allikrein by treating kallikreLn solutions with a carrier-bound water-insoluble pro~ease inhibitor which does not bind to kallikrein.
In this simple and elegant manner, highly pure kalli-krein solutions can be prepared which are s~able for longer periods than conventional ksllikrein solutions withou~ having to add stabilizers and without first having to lyophilize the kallikrein and dissolving it, only shortly before use, to form a suitable isotonic solution.
Suitable inhibitors for use in the present invention are, in particular, naturally occurring inhibitors having a polypeptide structure, for example a mixture of different protease inhibitors from soya bean or ~rom potato, or the purified trypsin inhibitor from soya be~n (KunL~z) or purified pro~ease inhibitors from potato or ~he ovomucoid inhibitor from hen's egg.
In particular, the protease inhibitQr mixture from potato is an easily accessible material which is very suitable for the purpose according to the invention.
With the aid of the process according to the invention9 it is in principle also possible to remove proteases from solution6 of nstural substances other than kallikrein; for this purpo~e, in additiGn to the inhibitors already mentioned, the Kunitz trypsin inhibitor from cattle organs is, in particular, ~lso suitable.
Such other solutlons, to which the process according to the invention can be applied analDgously are, in particular, ~5 solutions of enzyme~, for example asp~rRginase, carboxypep~ida A and B, zymogens, peptide hormone~, for example oxytoc;n, vasopressin, insulin and glucagon, as well as blood plasma and purified fractions prepared therefrom.
The above-mentioned inhibitors are bound to water-insoluble carriers in accordance wi~h general processes known to those skilled in the art. However, in each individual case the optimum conditions must be determined by experiment.
For example, the inhibitors mentioned, or further lnhibitors with similar properties, can be bound, according to German Published Specification 1,907,365, ~o cross-linked agarose, such as "Sepharose" ~ , by first activating the latter with BrCN. In the same way, the inhibitors can also be bound to other carr~ers containing hydroxyl groups, for example to cellulose or cross-linked dextran, ~uch as "Sephadex" ~ .
Alternetlvely, it is possible first to bind a low molecular weight amino compo~nd with BrCN to one of the above-mentioned carriers 80 that a derivative having a free amino or carboxyl group i8 produced, to which the inhibitor i8 bound by means of a water-soluble carbodiimide. The low molecular amino compound used for this purpose is, for example, 1,6-diaminohexane or 6-aminocaproic acid.
Furthermore, the inhibitors can be bound to polymers contalning carboxylic acid anhydrides, for example to a cnpolymer of tetraethylene glycol dimethacrylate and maleic snhydride according to Germ~n Publi~hed Specification 2,215,539,or a copolymer of tetraethylene glycol dimethacrylate, maleic anhydride 1(~53b~l and a hydrophilic monomer, for example methacrylic acid~ according to German Published Specification 2,215,687.
Further processes which can be used for the preparation of water-insoluble c~rrier-bound inhibitors have been summarized, for example, by P. Cuatreeasas and Ch. B. Anfinsen (Annual Reviews of Biochemistry, 1971).
The treatment,according to the inventio~ of the hlghly purified, optionally crystallized, kallikrein, prepared according to German Published Specification 27154,557 Gr other suitable processes, with the carrier-bound inhibitors is preferably carried out in dilute aqueous solution containing about 1 to about 50,000 KU/ml o~ kallikrein at ~ pH ~alue of about 5.0 to about 8Ø The solution can contain the usual neu~ral salts or buffer salts in concentrations of up to about 1.0 M, for ex6mple alkali metal or ammonium chloride~, ace~ates, formstes, carbonstes, citra~es, ph~sphates and the like. Pr~ferably, the treatment is carried ou~ in a salt-free solution or an opt~onally isotonic solution containing NaCl or ~odium acetate, 80 that the stabilized solution can be used directly, without desalination, for the productiDn of therapeutic preparations. The solution can furthermore contain suitable added preservatives, for example ethyl-mercuric thiosalicylate or benzyl alcoholO
The treatment of the aqueou~ kallikrein solution with the carrier-bound inhibitor may be carried out by stirring the carrier-bound in~ibitor into the kallikrein sclution and removing it, after a reaction time of at least about 10 minutes, by filtrati~n or centrifuging~ However, the method wherein the 1 ~5 3 ~ ~
carrier-bound inhibitor is introduced into a chromatography tube and the kalli~;rein solution is allowed to run through it is preferred.
The requisite amount ~f c~rrier-bound inhibitor depends on the inhibi~or l~sed and the degree of protease contamination of the solution to be treated. The carrier-b~und inhibitors can be regenerated by treatment with dilute acids or with acid buffer~, and can be used several times.
The success of the treatment of the kallikrein solution with carrier-bound inhibitors is shown by the reduced protease conten~ and the improved stability.
The protease content is measured by titratLng the aminoacid carboxyl groups liberated by the action of the protease-containing kallikrein solution on casein. A 6% strength solution ~f ca~ein (according to Hammersten, Merck 2242) in 0.1 N KCl i8 used and the titration is csrried out with 0.02 N RDH at pH 9.5 and 30C. 1 protease unit is defined as the amount of enzyme which splits 1 micro-equivalent of peptide bond in 1 minute under the conditions indicated.
In order to reduce the period of observation, the stab;lity of the kallikrein preparations was tested at 40C.
The kallikrein activity is determined by measuring the hydrolysis of N-benzoyl-~-arginine ethyl ester as a titrimetric test in the embodiment standardized by the F.l.P. ~Federation Internationale Pharmaceutique). 1 F.I.P. unit is def ined as the amount of kallikrein which splits 1 micromol of N-benzoyl-L-arginine ethyl ester in one minute at pH 8.0 and 25~C. The calculation to 1053~
convert the result to the customary kallikrein unit~ (KU), acc~rding to the de~inition of Frey, Kraut and Werle, is made by multiplying by a factor of 6.37.
The kallikrein solutions prepared by the process according to the invention can, in a manner which i~ in itself known and in dosages which are in themselves known, be employed directly as vasodilators and be administered by intramuscular or intravenous injection for treatment of peripheral vascular and coronary artery disea~e. The pro~ease-free stable l~llikrein solutions according to the invention are conveniently stored in the form of ampoules.
The present invention is illustrated by the drawing, which is a series of graphs showing the stability of various kallikrein preparations from the Examples which follow. The activity of each l~llikrein preparation, in per cent of the initial activity (_ 100), i8 plotted on the ord~na~e and ~he storage time of the kallilcrein preparations, at 40C in days, Ls shown on the abscissa. The individual curves in the drswing denote the following:
Curve 1: treated kallikrein from Example lb Curve 2: treated kallikreLn from Example 6 Curve 3: treated kallikrein from Example Sc Curve 4: treated kallikrein from ~xample 3b Curve 5: untreated kallikrein from Ex~mple 2 Curve 6: treated kallikrein from Example 10 Curve 7: untrea~ed k~llikrein from Examples 1 and 3 to 10.

1~?53~1 The invention is explained in more detail by the Examples which follow9 but is not restricted to the procedure~
presented in the Examples. All the kallikrein unit~ (KU) indicated Ln the Examples which follow are units according to Frey, Kraut and Werle.

Example 1 a) Binding soya trypsin inhibitors to"Sepharose".
10 ml of "Sepharose" 4 B (Pharmacia), a cross-l~nked agarose, were washed with water, suspended in 20 ml of water and activated for 5 minutes wLth 550 mg of BrCN in 20 ml of w~ter at pH 11 to 11.5. The product was filtered off on a glass frit and briefly washed with ice-cold 0.1 M NaHC03 solution. The activated "Sepharose" was immediately suspended Ln 30 ml of cold 0.1 M NaHC03 solution and 100 mg of soya trypsin inhibitor (highe~t purity, Serva) were added. The mixture was 6tirred for 24 hours at about 4C. The carrier-bound inhib~tor was then filtered off on a frit and wa~hed with 0.1 M borate of pH 8.0, 0.1 M acetate of pH 4.0 and water.
Spectrophotometric determlnation of the non-bound inhibitor in the filtrate and wash water indLcated 94 mg had been bound.

b) Treatment of kallikrein with carrier-bound soya trypspin inhLbltor.
50 mg of kallikrein having ~ specific activity of 1,013 KU/mg and protea~e cont~nt of 0.065 U/mg w~re dLssolved in 10 ml of H20 and filtexed ~hrough a column (0.9 x 15 cm~

1 05 3~ ~ 1 containing 10 ml of the ~arrier-bound lnhibitor prepared ~ccording to Example la. The filtrate contalning kallikrein was lyophllized and gave 49 mg containing 1,010 KU/mg (~ 97.5Z of ~heory~ and havLng a protease content of 0.005 U/mg.
The comparison of the stability of s~arting material (curve 7) and treated produet (curve 1) 18 shown in the drawing.

Example 2 700 mg of kallikrein having a specific activity of 1~080 KU/mg and a protease content of 0.058 U/mg were dissolved in water and riltered through 22 ml of a carrier-bound soya trypsin inhibitor prepared according to Example la. ~fter freeze-drying, the filtrate gave 695 mg having a specific activity of 1~050 KU/mg (_ 95Z of ~heory) and a prot~ase content of 0-005 U/mg.

~
fl) Blnding ovomucoid inhibitor to "Sepharose".
Following the procedure of Ex~mple la~ 10 ml of "Sepharose" were actLvated with BrCN and reacted with 100 mg of ovomucoLd (Worthington). 78 mg of ovomucoid were bound.

b) 50 mg of kallikrein (specific activity: 1~013 KU/mg;
protease: 0.065 U/mg) were di~solved in 0.02 ammonium acetate of pH 6.0 ~nd the solution was filtered through & column (0.9 x 15 cm) containLn& 10 ml of the carrigr-bound inhibitor prep~red aecording ts Example 3a. The kallikrein yield in ~he filtrate was g2X of theor~. ~ sample was de~alinated over "Ssphadex G-25" and ~0 5 3~ ~ ~
lyophilized. Specific activity: 980 KU/mg; prot~a~e: 0.008 U/mg.
Curve 4 shows the stability of the treated kallikrein and curve 7 the untreated.

Example 4 a) Prep~ration of inhibitor mixture from potato.
Washed potatoes were comminuted and suspended in a mixture of 0.95 kg of methanol, 0.375 kg of distilled water and 0.025 kg of 6070 strength perchloric acid per kg of potato.
Undissolved material was separated off on a filter press. The perchloric acid was neutralized by addition of pota~sium carbonate until the pH is 5.5. Potassium perchlorAte which had precipitated was filtered off. The filtrate was concentrated by evaporation in vacuo, then dialyzed and finally freeze-dried. About 450 ~g o~
inhibitor/kg of potato were obtained.

b) Binding of inhibitor mixture from po~ato to "Sepharo~e".
160 mg of an inhibLtor preparation according to Example 4a were reactedJ analogously to the procedure of Example la, wL~h 40 ml of BrCN-activated "Sepharose". 139 mg of inhibitor were bound.

c) 50 mg of kallikrein (specific actlvity 1~013 KU/mg;
protease: 0.065 U/mg~ were dissolved in 0.05 M ammonium acetate of pa 6.0 and the solution w~ f~ltered through ~ column ~0.9 x 15 cm) containing 10 ml of the carrier-bound inhibitor pxepared according to ~xample 4b. The knllikrein yield in the filtrate was 897o of theory. ~ de~alin~ted and lyophilLzed sample hsd a specific activity of 1~010 KU/mg and a protease content of 0-004 U/mg.

~5 3 ~ 1 Example 5 a) Preparation of purified protease inhibltor from potato.
80g of a protease inhibitor mixture from potato, prepared according to Example 4a, were applied to a column (9.~ x llO cm) containing 7 1 of carb~xymethyl-"Sephadex" C-50 in 0.01 M ammonium acetate of pH 4.5. The column Wa8 then first washed with 2 1 of the initial buffer and thereafter eluted with a linear concentration gradient of up to 1 M ammonium acetate of pH 5.4. Fractions of high inhibitor activLty ~gainst pancreas proteases were combined, concentra~ed, dialyzed and freeze-dried.
An enriched inhlbitor preparation was obtained in about 12% by weight yie~d relative to the crude preparation employed, or 55 mg/kg of potato.

b) Binding of purified proteace inhibitor from potato to "Sepharose".
160 mg o~ a purified inhibitor prepara~ion from potato were reacted with 40 ml of "Sepharose" in the same manner a~
described in Example 4b. 146 mg of the inhibitor were bQund.

c) In the same manner as in Example 4c, S0 mg of kallikrein were treated with 10 ml of the carrier-bound inhibitor prepared accDrding tD Example 5b. The yield was 95% ~f theory, the specific activity was 1~010 KU/mg and the protease c~ntent was 0.009 U/mg. Curves 3 and 7 compare the storage stability o~
the treated and untreated kallikrein.

1~536~1 ~.
4 ml of each of the carrier-bound inhLbitors prepared according to Examples lb, 3b and 5b were mixed and 50 mg of kallikrein were treated with the mixture analogously to Example 4c. The yield was 96%, the specific activity was 1,000 KU/mg and the protease content was 0.007 Ulmg. Curves 2 and 7 compare the storage stability of the treated and untre~ted kallikrein.

Example 7 a) Binding of 1,6-dLaminohexane to "Sephsrose".
100 ml of "Sepharose" were actLvated with 15g of BrCN
znd then reacted with 23.3g of 1,6-diaminohex~ne. The product was wsshed w~th borate buffer of pH 8.5, acetate buffer of pH 4.0 and water. A freeze-dried sample had ~ nLtrogen eontent of 5.9~.

b) Binding of protease inhibitor from potato to "Sepharose"
sub~ti~uted with 1,6-diam~nohexsne.
16 ml of the Sepharose derivative according to Ex~mple 7a were suspended in 15 ml of water and stirred, after ~ddition of 200 mg of protease inhibitor from potato obtaLned from Example 5a and 200 mg of ethyl-(3-dimethylamino-propyl)-carbodi-imide hydrochloride for 24 hours ~t pH 4.7. The product ~as then flltered off and washed with 1 M N~Cl and water. 189 mg of inhibitor were bound.

~ ~ 5 3~ 1 c) 50 mg of kallikrein were treated, as described in Example 4~, with the carrier-bound lnhibitor prepared according to Example 7b. The yield wa~ 93% of theory, the spec~fic activity 995 KU/mg and the protease content 0.011 U/mg.

Ex~mple 8 a) Binding of protease inhibitor from potato to "Sepharose"
substituted with 6-aminocaproic scLd.
16 ml of "Sepharose" substituted with 6-aminocaproic acid (CH-"Sepharose") ~sre r2acted with 200 mg of protease inhibitor from potato as described in Example 7b. 195 mg of inhibitor were bound.

b) 50 mg of kallikrein were treated, as described in Example 4c, with the carrier-bound inhibitor prepared according to Example 8a. The yield was 9270 of theory, the specific activity 988 KU/mg and the protease content 0.0095 U/mg.

a) Preparation of a copolymer cf tetraethylene glycol dimeth-acrylate, methacrylic acid ~nd maleic anhydride.
60g of tetraethylene glycol dimethacryla~e, 30g of methacrylic acid, lOg of maleic anhydride and lg of azoiso-butyronitrile are dissolved in 300 ml of acetonitrile. Thi~
solution i~ suspended in 1 1 of benzene (boiling poLnt 100-140C), which contains 5g of ~ mixture of glycerol monooleate and glycerol dioleate, and polymeri~-ed for 22 hours at 60C.

~(~5 ~
The polymer beads are filtered off, suspended three times in benzene and twice in petroleum ether (boiling point 30-50C) and dried in vacuo.
Yield: 94g Bulk volume: 44 mlfg Swelling volume in water: 5.5 ml/g Specific surface area: 6.6 m2lg Acid content after saponification of the anhydride groups:
4.3 milliequivalents/g.

b) Binding of pr3tease inhibitor from potato.
lOg of a copolymer according to Example 9a were washed with 100 ml of scetone and then suspended in 880 ml of water.
40 ml of 0.1 M triethylamine were added and the pH w s ad~u~ted to 6.2 with 0.1 M acetic acid in exactly 1 minute. 1.0g of prot~ase inhibitor from potato according to Example 5a wa~ then Added hnd the pH was kept at 6.2. After 24 hours, the carrier-bound inhibitor was filtered off and washed with 0.1 M sodium borate of pH 8.5, 0.1 M sodium acetate of pH 4.0 and water.
280 mg of inhibitor were bound.

c) 50 mg ~f kallikrein were treated~ as de~eribed in Example 4c, with 10 ml of the carrier-bound inhibitor prepared according to Example 9b. The yield was 88% of theory, the specific activity 1~005 KU/mg and the protease content 0.011 U/~g.
The results of the treatment of kallikrein with the various carrier-bound inhibitors are summarized in the Table which follows.

~5 3 IABLE

Result of the treatment of_kallikreLn with carrier bound inhibitors Example Start ng material Tre~ted kallikrein KU/mg Protease-U/mg Yield, % KU/m$ Protease-U/mg ._ "
lb \ \ 97.51~010 0.005 3b ~ ~ 92 980 0.008 4c / 1 89 l)010 0.004 5c~1~013 ~ 0.065 95 1,010 0.009 6 96 1)000 0.007 7c 93 995 0.011 8b . 92 988 0.0095 9c J 88 1~005 0.011 ,.,_ . ,.-.. _._ ; .,_ ._ .- ~ - - .. _ 21~080 0.05~ 95 l~OOS 0.0~5 53b~l Example 10 - ComPaxison Example Chromato~raphY of hi~hlY purified kallikrein on DEAE-SePhadex A-50 100 mg of kallikrein having a specific activity of 1~013 KU/mg and a protease content of 0.065 U/mg were dissolved S in 50 ml of sodium acetate buffer of pH 5.0, 12 mS/cm and charged onto a 2.5 x 40 cm column of DEAE-Sephadex A-50 in the same buffer. The column was rinsed with 300 ml of sodium acetate of pH 5.0, 14 mS/cm and eluted with a linear gradient of 300 ml each of sodium acetate pH 5.0 14 mStcm and 0.5 M respectively.
The active fractions of the eluate (210 ml) were combined, concentrated to 20 ml by ultrafiltration, desalinated on a column of Sephadex G-25 and freeze-dried.
Yield: 61 mg with 1~320 K~/mg _ ~9Z
Pro~ea~e content: 0.014 U/mg The s~bility of this preparation i8 shown ~8 curve 6 in the drawing.

Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN
EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE
DEFINED AS FOLLOWS:

1. A method of removing protease from kallikrein comprising contacting a solution of the kallikrein with a carrer-bound wanter-insoluble protease inhibitor that does not bind to said kallikrein and recovering protease-free kallikrein.

2. A method according to claim 1, wherein the protease inhibitor is a potato, soya bean, or ovomucoid protease inhibitor.

3. A method according to claim 2, wherein the protease inhibitor is bound to a cross-linked agarose, cellulose, a cross-linked dextran, or a polymer containing carboxylic anhydride groups.

4. A method according to claim 1, wherein the solution is a salt free or blood-isotonic solution.

5. A method according to claim 1, wherein an aqueous kallikrein solution containing from about 1 to about 50,000 KU/ml at a pH of from about 5.0 to about 8.0 is contacted with said carrier-bound protease inhibitor.

6. A method according to claim 5, wherein said aqueous solution is salt-free or blood-isotonic.

7. A method according to claim 5, wherein the carrier-bound protease inhibitor is in a chromato-graphy tube and said aqueous solution is introduced into said tube and allowed to run through the tube.

8. Protease-free kallikrein whenever prepared by the method of claim 1, 2 or 3.

9. Protease-free kallikrein whenever prepared by the method of claim 4,5, or 6.

10. Protease-free kallikrein whenever pre-pared by the process of claim 7.

11. A method of removing protease from kallikrein comprising contacting a solution of the kallikrein with a carrier-bound water-insoluble protease inhibitor that does not bind to said kallikrein, and recovering kallikrein having an activity of about 1000-1300 KU/mg and a protease content of not more than about 0.011 U/mg.

12. Kallikrein having an activity of about 1000-1300 KU/mg and a protease content of not more than about 0.011U/mg whenever prepared by the process of claim 11.

13. The method according to claim 11, wherein the recovered kallikrein has a protease content of about 0.004 to about 0,011 U/mg.

14. Kallikrein having an activity of about 1000-1300 KU/mg and a protease content of about 0.004 to about 0.011U/mg whenever prepared by the method of claim 13.

15. The method according to claim 13, wherein the kallikrein solution is a salt-free or blood-isotonic aqueous kallikrein solution containing from about 1 to about 50,000 MU/ml at a pH of from about 5.0 to about 8Ø

16. Kallikrein in the form of a salt-free or blood-isotonic aqueous solution containing from 1 to 50,000 KU/ml of kallikrein, said kallikrein having an activity of about 1000 to 1300 MU/mg and a protease content of about 0.004 to about 0.011 U/mg, whenever prepared by the method of claim 15.