CA1040562A - Methods for purifying urokinase preparations - Google Patents
Methods for purifying urokinase preparationsInfo
- Publication number
- CA1040562A CA1040562A CA240,777A CA240777A CA1040562A CA 1040562 A CA1040562 A CA 1040562A CA 240777 A CA240777 A CA 240777A CA 1040562 A CA1040562 A CA 1040562A
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- solution
- urokinase
- micromhos
- ammonium sulphate
- proteins
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Abstract
ABSTRACT OF THE DISCLOSURE
A method of purifying a urokinase solution mixed with foreign proteins and pyrogenic substances, in which the aqueous urokinase solution is partially saturat?d with an agent which precipitates proteins to an extent, depending on the initial protein concentration of the solution, such that the partial sa-turation precipitates only part of the proteins in the initial solution, the part being sufficiently small for its urokinase content to be kept within predetermined proportions which are small or practically negligible, and the supernatant liquid containing the urokinase is collected. The purified urok-nase so obtained can be converted in to urokinase hepavinate, which is useful in the treatment of thrombo-embolic syndromes.
A method of purifying a urokinase solution mixed with foreign proteins and pyrogenic substances, in which the aqueous urokinase solution is partially saturat?d with an agent which precipitates proteins to an extent, depending on the initial protein concentration of the solution, such that the partial sa-turation precipitates only part of the proteins in the initial solution, the part being sufficiently small for its urokinase content to be kept within predetermined proportions which are small or practically negligible, and the supernatant liquid containing the urokinase is collected. The purified urok-nase so obtained can be converted in to urokinase hepavinate, which is useful in the treatment of thrombo-embolic syndromes.
Description
~L04~i6Z ;:? .
This invention relates to a method for the purification and particular-: -c,, ly the depyrogenation (i.e. the elimination of pyrogenic substances) of uroki- ~
,,, .:, .
nase. More specifically, the invention relates to a method purifying urokinase ' ~
;~., ~ .. , preparations obtained from biological media~ such as for example, urine of ani-mal origin and particularly human origin.
It is known that urokinase is an enzyme present in urine and which catalyses the transformation of the plasminogen into plasmin, of which pepti-.
dase the physiological role is to dissolve intravascular fibrinous formation.
A fault in the mechanisms of activation of the plasminogen, in the case of ac- `~
celerated kinetics of the coagulating system or of alteration of the vascular walls, leads to the maintenance of intravascular fibrin which in the end pro-vokes various thrombo-embolic syndromes (thronlbosis, emoblisms, disseminated ~ ;
intravascular coagulation, etc.). The administration of urokinase in these pathological cases causes acceleration of the formation of plasmln, especially at the level of fibrinous formations, as indicated in the literature.
,:, , ,: , Urokinase therefore constitutes the preferred treatment of thrombo- ~ ~
embolic syndromes and its human origin avoids the dangers of anaphylactic shock. !;~,~,,, There have already been proposed a certain number of methods for ob- ~ ;
taining purified preparations of urokinase, which methods however involve, ge-nerally, relatively high losses in yield of urokinase. These losses in yield . ,i~ ,, .
can hence be all the more ~mdesirable as the production costs of purified uro~
kinase are higher and, in the case of urokinase extracted from human urine, as the collection of the latter in large amounts is difficult.
;.: : - .
In addition known purification methods do not generally allow the pro-duction of urokinase preparations whose content of pyrogenic substances is suf-ficiently reduced for their use in therapeutics to be easy. In fact this con-tent of pyrogenic substances is of such a nature as to constitute a drawback j ;
which can become serious if account is taken of the extreme sensitivity often observed to arise in patients from treatment based on urokinase, which treat-ment involves the administration by the intravenous route of doses of the i -1- ~ ; , ':
i .
;,,i,' :~. " :' . " .
``; 1041~56Z `
urokinase which can be high, capable, for example, of reaching 150,000 CTA
units or more in a single injection. (CTA units are standard units of uroki-nase activity adopted by the Committee on Thrombolytic Agents, National Heart Institute, U.S.A.) According to the present invention, there is provided a m~thod of purifying a urokinase solution mixed with foreign proteins and pyrogenic sub-stances, in which the aqueous urokinase solution is partially saturated with -an agent which precipitates proteins to an extent, depending on the initial ., protein concentration of the solution, such that the partial saturation pre-cipitates only part of the proteins in the initial solution, the part being ~ ;
sufficiently small for its urokinase content to be kept within predetermined ;~
proportions which are small or practically negligible, and the supernatant liquid containing the urokinase is collected.
Par~icularly ~he present invention provides a method of purifying a urokinase solution mixed with foreign proteins and pyrogenic substances, which comprises partially saturating the solution with ammonium sulphate up :
to a degree which causes a minor proportion, not exceeding 5%, of the total activity of the urokinase in the initial solution to be precipitated with v part of the proteins initially contained in said solution and collecting the supernatant liquid containing the urokinase substantially freed from pyrogenic substances.
Advantageously, the urokinase solution treated in accordance with the method of the invention is an enrichecl urokinase preparation obtained from , human urine by a method involving the following two steps:
the first step comprises subjecting the above mentioned concentrated solution to exclusion chromatography by placing it in contact with a diethyl-aminoethylcellulose resin, known as DEAE cellulose, the conductivi~y of the solution having previously been adjusted to from 15000 to 25000 micromhos/cm and the pH to from 4 to 6 and preferably about 4.5. The effluent obtained is collected and, preferably, that the resin is washed with a solution of ~, ~' ' ,:' ., . , ` ' `, ~04(~S6Z :
ammonium sulphate at pH 4.5 and of which the conductivity is of the order of 15,000 micromhos per centimeter;
the second step, which is preferably carried out on an initial solution resulting from the combination of the effluent and the washings .
: obtained at the end of the above said first step, comprises subjecting this solution to a second exclusion chromatography by placing it in contact with .
. a diethylaminocellulose resin. Prior to this placing in contact, on the one : hand, the conductivity of the solution is adjusted to a value at least equal . to 15,000 , ' ':
, ''' ,', :.
" .
,,: i ~ .. ..
' ' ., ' '' `.. -.: ' ,' . . .
`~' ;,~, .` ', ' ': .
.:' ' .
""
','':, - 2a -. . .
micron~os per centimetre, preferably comprised between 15,000 and 18,000 micromhos per centimetre, and its pH to a value comprised between 6 and 7, pre-ferably comprised between 6.6 and 6.8 and, on the other hand, the weight of resin used in this second exclusion chromatography is adjusted with relation to the urokinase activity of the solution to a sufficiently high value for it to be possible to observe a retention of proteins on the resin under the above-. . .,: .
; indicated conditions of conductivity and of p~, this value not however-exceed-ing that for which there would also be observed a non-negligible retention of ;
urokinase on the resin; the conductivity of the solution in contact with the ;
resin being then, during the above-said second exclusion chromatography, brought by successive steps, by addition of apyrogenic distilled water, to a final value of the order of 9,000 micron1hos.
Advanta~eously, each addition of water is such that it produces the ' lowering of conductivity of the medium by a value of the order of 1,000 mi-cromhos per centimetre, each addition being separated by a shaking operation during a time comprised between 5 and 15 minutes, preferably of the order of il 10 minutes. ~ ''' All the preceding operations are, preferably, effected at a tempera-ture of the order of 4C.
It will be observed that the invention has thus proposed conditions in which preferential absorption of the proteinic impurities contained in the concentrated solution of urokinase occurs, the latter not itself being retained `
on the resin. The principal parameter playing part in the establishment of these differential absorption conditions, is constituted by tne conductivity of the treated solutions.
Respect for the ranges of conductivity, u~der the pH conditions indi-cated above, is essential for the good operation of the method according to the invention and for the obtaining of high yields of urokinase. It will be noted in particular that, in the case of the first exclusion chromatography~ the . 30 resin can fix neither urokinase, nor proteinic impurities if the initial con-_3_ '~. , ', . ' . , : , . , :
f ~4~)562 ~
~ ductivity of the concentrated solution of urokinase is higher than 25,000 . , .
micromhos per centimetre. It absorbs on the other hand at the same time uro-kinase and the proteinic impurities if the conductivity of the concentrated ~`
solution of urokinase is adjusted to a value below 10,000 micromhos per cen- ~
timetre. ~ -It is the same at the stage of the second exclusion chromatography. ~
.. .. ...
For initial conductivities of 15,000 to 18,000 micromhos of the treated solu- ~
'7 '' ' ' tion there is observed a very selective absorption of the proteins. ~ach of the successive dilutions results in an additional absorption of proteins on the ; ~ ;
resin. The gradual saturation of the latter involves a reduction in its affi- `
nity for urokinase, so that the above-said successive dilutions can be pursued to a value of 9,000 micromhos per centimetre, without risk of also absorbing urokinase resulting therefrom.
Similarly it is important, especially in the second exclusion chro-matography to keep the values of the ratio of the amount of resin applied to the urokinase activity of the treated solution between certain limits. l This ratio can vary within rather wide limits in the first exclusion ~`
chromatography. It has been observed in particular that similar results are obtained for all values of this ratio comprised between about 15 g and 40 g of dry resin per 14 million CrA units of urokinase.
The selection of this ratio is more critical in the case of the se-cond exclusion chromatography. It must be kept, especially for the values of tlle conductivity located around 22,000 micro~los per centimetre, between about 30 and about 60g of dry resin per 10 million CTA units of urokinase of the ~ :
solution to be treated, the best results being obtained for ualues comprised between 40 and 53 g of dry resin per 10 million CTA unlts of urokinase.
. .
In certain cases the concentrated solution of urokinase can be sub-jected directly to the above-said second exclusion chromatography, especially !, in the case where recourse is had, in the steps of final purification, to special subsequent steps of separation of the pyrogenic substances which, in , .. . :. . .~ : ,. . . ;~ : : . : .
~ 401S6Z
human urine, always accompany the urokinase. In practice it is however most-ly preferable to start with the above-said first exclusion chromatography.
The latter enables in fact, besides a first elimination of a small amount of -protein impurities, the obtaining of a perfectly clear solution and a first depyrogenation of the urokinase.
A considerable enrichment of the urokinase relative to the proteins which initially accompany it is so obtained, 50 to 60% of the proteins being removed, with a total yield of urokinase activity.
The solution thus enriched in urokinase can again be further puri-fied by having recourse to additional treatments known in principle, espec-ially by absorption of the solution of kaolin, then by absorption chromato-graphy on resin of the Amberlite* type or again by absorption chromatography on carboxymethyl cellulose.
It is thus possible by this method, completed by one of the techni-ques which have been described and applied to a solution of crude urokinase extracted from human urine finally to remove 80-90% of the proteins which initially accompany the urokinase, without notable losses of urokinase activity.
The concentrated solution of crude urokinase initially applied can be obtained in any manner known in itself, for example in the case of the ex-traction of urokinase from very dilute solutions, such as urine, especially human urine, by placing the latter in contact with an absorption agent for the proteins and by elution of the absorbate. These are described in the literature various methods enabling the production of initial concentrated solutions of crude urokinase, these methods utilising absorption agents as diverse as, for example, silicate gel, cation exchange silicates, salts such as barium sulphate, resins such as oxycellulose, etc.
Advantageously, recourse will be had to the placing of the solut-ions in contact with a filtration adjuvant such as that marketed under the trademark "Llyflosupercel", this operation being followed by re-extraction of the urokinase and of the proteins absorbed by the filtration adjuvant, by a :~ .
.. .
~ ~ , *Trademark ~5~
::!
f- :
~0gL~562 salting out of the urokinase contained in the extraction solution by means of ; ammonium sulphate, by solution of the precipitate obtained in a glucose solu-tion and by dialysis of the latter to obtain finally a dialysate which is sub-jected to one of the exclusion chromatography steps, preferably the first, of the method according to the invention. ~
This content of pyrogenic substances in the enriched solutions so -:
; obtained, is not however yet negligible. In fact such purified preparations ; of urokinase still produce a certain hyperthermia in the rabbit when the doses administered exceed a moderately high threshold. Injected in rabbits ., .
under the conditions according to the general methods of analysis of the French Pharmacopea, 8th edition, page 1615, at the dose of 3000 CTA units/kg, these purified preparations of urokinase produce an elevation of temperature .
which varies from 0.5 to 0.9C from one rabbit to the other.
In order to obtain preparations of urokinase having still further ~ , reduced contents of pyrogenic substances recourse is had to the method according to the invention of purification, more particularly of depyrogenat-ion, from proteinic substances with biological activity, especially urokinase, which preferably has been subjected previously to a first purification, this ", method being characterised in that a partial saturation of this solution is produced with an agent for the precipitation of the proteins to a degree, which is a function of the initial concentration in the solution of the pro-teins, such that this partial saturation only induces the precipitation of a portion of the proteins contained in the initial solution, this portion ~ being sufficiently reduced for its content in the proteinic substance with ; biological ac~ivity to be maintained in predetermined low proportions, if not practically negligible, and the supernatant liquid is collected.
In a preferred embodiment of the method according to the invention, applied to urokinase, the precipitating agent is constituted by ammonium sul-phate, the above-said partial precipitation being effected on a urokinase .
solution containing from 1.5 to 3 mg of proteins per ml, at a pH comprised ~' .~: ,~ , ,;, between 3 and 4.5, preferably of the order of 3.8, the conductivity of this :~ , .. .. .
~ solution having been adjusted previously to a value comprised between 15,000 ; and 25,000 micromhos per centimetre, the degree of saturation of the solution ;`
in ammonium sulphate, comprised between 0.25 to 0.35, not being pushed beyond ,; :
that which produces precipitation of proteins containing more than 5% of the urokinase activity contained in the initial solution ~being understood that ~-the degree of saturation of a solution equals 1, when the solution is comple-tely saturated: that is to say by the solution of 700 g of ammonium sulphate in 1,000 ml of distilled water at -~4C). The urokinase practically free of pyrogenic substances contained in the supernatant liquid can then be isolated, especially by ~alting the solution with crystalline ammonium sulphate.
It will be appreciated that, taking into account the preferred ranges of concentrations of proteins indicated above, it will be advantageous to pro-ceed with a preliminary puriEication as extensive as possible of the urokinases to be treated, to eliminate the maximum possible of proteins, so that it is not necessary to operate with too large volumes of solution. In other words, the method according to the invention constitutes, in one of its preferred applica-tions, a final purification step intended to perfect the effects of a prelimina-ry purification method. ~`
Advantageously recourse will be had to the method of purification cles~;ribed above, comprising the two above-indicated exclusion chromatography steps, completed by a third step comprising:
either an absorption chromatography on carboxymethyl-cellulose of the urokinase contained in the solution resulting from combination of the effluent and the washings from the resin of the second exclusion chromatography, at a pH of the order 5, and the elution of the urokinase absorbed on carboxymethyl-cellulose with a phosphate buffer at pH 6.8 - 6.9, or an absorption of the solution resulting from the conibination of the effluent and the washings of the second exclusion chromatography on kaolin at pH 6.2, the elution of the urokinase held on the kaolin by a solution of ;1`';. ' '.,: '.
. ~L04~562 ammonium chloride in 4% ammonia, precipitation with ammonium sulphate, taking ,: -; up the precipitate in water an-l dialysis of the suspension against an 0.05 M
phosphate solution buffered to p~ 6.2, filtration of the dialysate on a column of resin of the Amberlite IRC 50 type, and elution of the resin to recover the urokinase.
It has been noted, as already indicated above, that the degree of saturation of the solution in the ammonium sulphate wnich produces the forma~
tion of a precipitated protein, (among which are the pyrogenic substances) retaining a given minimum percentage of urokinase varies as a function, espe-cially inversely, of the initial content of prokeins of the treated solution.
The values which are suitable to be given to this degree of saturation, taking into account the initial content of the solution in proteins, will be .apparant especially from the graph of the single Figure of the accompanying drawings. These values have been cletermined experimentally, especially on solut:ions purified according to the preferred method of purification indicated below, in particular on those obtained in the Examples described below rhe axis A of the single figure is graduated in concentrations of proteins (in mg/ml) of solutions to be treated, the C axis in degrees of saturation (S) of the solution of ammonium sulphate and the B axis in precent of urokinase en- '!
trained in the precipitated proteins formed under the conditions of the inven-tion. The relative arrangements of these axes and the scale of thier gradua-tions have been selected so that the percentage of urokinase entrained is, in ; each case, given by the point of intersection with the B axis of a line passing through the points representing the concentration in proteins, on the A axis, and of the degree of saturation in am~nium sulphate on the C axis respectively. .:
Thus there is obtained, for example, a protein precipitate holding 5% of the urokinase activity of the initial solution, by bringing to 0.30 the degree of saturation in ammonium sulphate of the solution, when the initial content in proteins of the latter is 2 mg/ml (dashed line a), or by bringing it to 0.25 when this initial content is 3 mg/ml ~mixed line _). `:
.. , . . , : :,. ., , . . . .. ~ . :
lO~S62 Considering for example3 the case of an initial solution of urokinase containing 2mg/ml of proteins, in more specific manner, the following observa-tions may be made.
Up to a value of the degree of saturation equal to 0.20, there is no formation of precipitate. The solution obtained hence conserves its pyrogenic substances. At the same concentration of proteins and with a degree of satura-tion equal to 0.25, a slight precipitate begins to be formed, this precipitate retaining about 2.5% of the urokinase contained in the medium. This saturation is not sufficient to retain the whole of the pyrogenic substances. On the other hand, with a degree of saturation of 0.30, the precipitate formed which contains about 5% of urokinase, retains also the larger part of the pyrogenic substances. The supernatant liquid, which still contains 95% of the total urokinase activity contained in the initial solution, is free of pyrogenic sub-stances according to the standards fixed by the French Pharmacopea already men-tioned, at doses as high as 15,000 CTA units/kg. At a saturation above 0.30, the supernatant liquid will also be a pyrogenic at the same doses but the amount of urokinase retained on the precipitate will be greater, as shown in the graph.
Additional characteristics of the invention will become apparent also in the course of the description of the ~xamples which follow. It is under-stood that in the following, all the operations are carried out at a tempera-ture of the order of 4C, except in the case where a different temperature is expressly stipulated.
Fxample I - Preparation of purified urokinase One thousand litres of urokinase are collected on phenol. The amount of phenol utilised for this purpose is such that at the end of the collection, the concentration of phenol is at a minimum 0.5%. By the addition of acetic acid, the p~ of the urine thus collected is adjusted to 5.8. ~-
This invention relates to a method for the purification and particular-: -c,, ly the depyrogenation (i.e. the elimination of pyrogenic substances) of uroki- ~
,,, .:, .
nase. More specifically, the invention relates to a method purifying urokinase ' ~
;~., ~ .. , preparations obtained from biological media~ such as for example, urine of ani-mal origin and particularly human origin.
It is known that urokinase is an enzyme present in urine and which catalyses the transformation of the plasminogen into plasmin, of which pepti-.
dase the physiological role is to dissolve intravascular fibrinous formation.
A fault in the mechanisms of activation of the plasminogen, in the case of ac- `~
celerated kinetics of the coagulating system or of alteration of the vascular walls, leads to the maintenance of intravascular fibrin which in the end pro-vokes various thrombo-embolic syndromes (thronlbosis, emoblisms, disseminated ~ ;
intravascular coagulation, etc.). The administration of urokinase in these pathological cases causes acceleration of the formation of plasmln, especially at the level of fibrinous formations, as indicated in the literature.
,:, , ,: , Urokinase therefore constitutes the preferred treatment of thrombo- ~ ~
embolic syndromes and its human origin avoids the dangers of anaphylactic shock. !;~,~,,, There have already been proposed a certain number of methods for ob- ~ ;
taining purified preparations of urokinase, which methods however involve, ge-nerally, relatively high losses in yield of urokinase. These losses in yield . ,i~ ,, .
can hence be all the more ~mdesirable as the production costs of purified uro~
kinase are higher and, in the case of urokinase extracted from human urine, as the collection of the latter in large amounts is difficult.
;.: : - .
In addition known purification methods do not generally allow the pro-duction of urokinase preparations whose content of pyrogenic substances is suf-ficiently reduced for their use in therapeutics to be easy. In fact this con-tent of pyrogenic substances is of such a nature as to constitute a drawback j ;
which can become serious if account is taken of the extreme sensitivity often observed to arise in patients from treatment based on urokinase, which treat-ment involves the administration by the intravenous route of doses of the i -1- ~ ; , ':
i .
;,,i,' :~. " :' . " .
``; 1041~56Z `
urokinase which can be high, capable, for example, of reaching 150,000 CTA
units or more in a single injection. (CTA units are standard units of uroki-nase activity adopted by the Committee on Thrombolytic Agents, National Heart Institute, U.S.A.) According to the present invention, there is provided a m~thod of purifying a urokinase solution mixed with foreign proteins and pyrogenic sub-stances, in which the aqueous urokinase solution is partially saturated with -an agent which precipitates proteins to an extent, depending on the initial ., protein concentration of the solution, such that the partial saturation pre-cipitates only part of the proteins in the initial solution, the part being ~ ;
sufficiently small for its urokinase content to be kept within predetermined ;~
proportions which are small or practically negligible, and the supernatant liquid containing the urokinase is collected.
Par~icularly ~he present invention provides a method of purifying a urokinase solution mixed with foreign proteins and pyrogenic substances, which comprises partially saturating the solution with ammonium sulphate up :
to a degree which causes a minor proportion, not exceeding 5%, of the total activity of the urokinase in the initial solution to be precipitated with v part of the proteins initially contained in said solution and collecting the supernatant liquid containing the urokinase substantially freed from pyrogenic substances.
Advantageously, the urokinase solution treated in accordance with the method of the invention is an enrichecl urokinase preparation obtained from , human urine by a method involving the following two steps:
the first step comprises subjecting the above mentioned concentrated solution to exclusion chromatography by placing it in contact with a diethyl-aminoethylcellulose resin, known as DEAE cellulose, the conductivi~y of the solution having previously been adjusted to from 15000 to 25000 micromhos/cm and the pH to from 4 to 6 and preferably about 4.5. The effluent obtained is collected and, preferably, that the resin is washed with a solution of ~, ~' ' ,:' ., . , ` ' `, ~04(~S6Z :
ammonium sulphate at pH 4.5 and of which the conductivity is of the order of 15,000 micromhos per centimeter;
the second step, which is preferably carried out on an initial solution resulting from the combination of the effluent and the washings .
: obtained at the end of the above said first step, comprises subjecting this solution to a second exclusion chromatography by placing it in contact with .
. a diethylaminocellulose resin. Prior to this placing in contact, on the one : hand, the conductivity of the solution is adjusted to a value at least equal . to 15,000 , ' ':
, ''' ,', :.
" .
,,: i ~ .. ..
' ' ., ' '' `.. -.: ' ,' . . .
`~' ;,~, .` ', ' ': .
.:' ' .
""
','':, - 2a -. . .
micron~os per centimetre, preferably comprised between 15,000 and 18,000 micromhos per centimetre, and its pH to a value comprised between 6 and 7, pre-ferably comprised between 6.6 and 6.8 and, on the other hand, the weight of resin used in this second exclusion chromatography is adjusted with relation to the urokinase activity of the solution to a sufficiently high value for it to be possible to observe a retention of proteins on the resin under the above-. . .,: .
; indicated conditions of conductivity and of p~, this value not however-exceed-ing that for which there would also be observed a non-negligible retention of ;
urokinase on the resin; the conductivity of the solution in contact with the ;
resin being then, during the above-said second exclusion chromatography, brought by successive steps, by addition of apyrogenic distilled water, to a final value of the order of 9,000 micron1hos.
Advanta~eously, each addition of water is such that it produces the ' lowering of conductivity of the medium by a value of the order of 1,000 mi-cromhos per centimetre, each addition being separated by a shaking operation during a time comprised between 5 and 15 minutes, preferably of the order of il 10 minutes. ~ ''' All the preceding operations are, preferably, effected at a tempera-ture of the order of 4C.
It will be observed that the invention has thus proposed conditions in which preferential absorption of the proteinic impurities contained in the concentrated solution of urokinase occurs, the latter not itself being retained `
on the resin. The principal parameter playing part in the establishment of these differential absorption conditions, is constituted by tne conductivity of the treated solutions.
Respect for the ranges of conductivity, u~der the pH conditions indi-cated above, is essential for the good operation of the method according to the invention and for the obtaining of high yields of urokinase. It will be noted in particular that, in the case of the first exclusion chromatography~ the . 30 resin can fix neither urokinase, nor proteinic impurities if the initial con-_3_ '~. , ', . ' . , : , . , :
f ~4~)562 ~
~ ductivity of the concentrated solution of urokinase is higher than 25,000 . , .
micromhos per centimetre. It absorbs on the other hand at the same time uro-kinase and the proteinic impurities if the conductivity of the concentrated ~`
solution of urokinase is adjusted to a value below 10,000 micromhos per cen- ~
timetre. ~ -It is the same at the stage of the second exclusion chromatography. ~
.. .. ...
For initial conductivities of 15,000 to 18,000 micromhos of the treated solu- ~
'7 '' ' ' tion there is observed a very selective absorption of the proteins. ~ach of the successive dilutions results in an additional absorption of proteins on the ; ~ ;
resin. The gradual saturation of the latter involves a reduction in its affi- `
nity for urokinase, so that the above-said successive dilutions can be pursued to a value of 9,000 micromhos per centimetre, without risk of also absorbing urokinase resulting therefrom.
Similarly it is important, especially in the second exclusion chro-matography to keep the values of the ratio of the amount of resin applied to the urokinase activity of the treated solution between certain limits. l This ratio can vary within rather wide limits in the first exclusion ~`
chromatography. It has been observed in particular that similar results are obtained for all values of this ratio comprised between about 15 g and 40 g of dry resin per 14 million CrA units of urokinase.
The selection of this ratio is more critical in the case of the se-cond exclusion chromatography. It must be kept, especially for the values of tlle conductivity located around 22,000 micro~los per centimetre, between about 30 and about 60g of dry resin per 10 million CTA units of urokinase of the ~ :
solution to be treated, the best results being obtained for ualues comprised between 40 and 53 g of dry resin per 10 million CTA unlts of urokinase.
. .
In certain cases the concentrated solution of urokinase can be sub-jected directly to the above-said second exclusion chromatography, especially !, in the case where recourse is had, in the steps of final purification, to special subsequent steps of separation of the pyrogenic substances which, in , .. . :. . .~ : ,. . . ;~ : : . : .
~ 401S6Z
human urine, always accompany the urokinase. In practice it is however most-ly preferable to start with the above-said first exclusion chromatography.
The latter enables in fact, besides a first elimination of a small amount of -protein impurities, the obtaining of a perfectly clear solution and a first depyrogenation of the urokinase.
A considerable enrichment of the urokinase relative to the proteins which initially accompany it is so obtained, 50 to 60% of the proteins being removed, with a total yield of urokinase activity.
The solution thus enriched in urokinase can again be further puri-fied by having recourse to additional treatments known in principle, espec-ially by absorption of the solution of kaolin, then by absorption chromato-graphy on resin of the Amberlite* type or again by absorption chromatography on carboxymethyl cellulose.
It is thus possible by this method, completed by one of the techni-ques which have been described and applied to a solution of crude urokinase extracted from human urine finally to remove 80-90% of the proteins which initially accompany the urokinase, without notable losses of urokinase activity.
The concentrated solution of crude urokinase initially applied can be obtained in any manner known in itself, for example in the case of the ex-traction of urokinase from very dilute solutions, such as urine, especially human urine, by placing the latter in contact with an absorption agent for the proteins and by elution of the absorbate. These are described in the literature various methods enabling the production of initial concentrated solutions of crude urokinase, these methods utilising absorption agents as diverse as, for example, silicate gel, cation exchange silicates, salts such as barium sulphate, resins such as oxycellulose, etc.
Advantageously, recourse will be had to the placing of the solut-ions in contact with a filtration adjuvant such as that marketed under the trademark "Llyflosupercel", this operation being followed by re-extraction of the urokinase and of the proteins absorbed by the filtration adjuvant, by a :~ .
.. .
~ ~ , *Trademark ~5~
::!
f- :
~0gL~562 salting out of the urokinase contained in the extraction solution by means of ; ammonium sulphate, by solution of the precipitate obtained in a glucose solu-tion and by dialysis of the latter to obtain finally a dialysate which is sub-jected to one of the exclusion chromatography steps, preferably the first, of the method according to the invention. ~
This content of pyrogenic substances in the enriched solutions so -:
; obtained, is not however yet negligible. In fact such purified preparations ; of urokinase still produce a certain hyperthermia in the rabbit when the doses administered exceed a moderately high threshold. Injected in rabbits ., .
under the conditions according to the general methods of analysis of the French Pharmacopea, 8th edition, page 1615, at the dose of 3000 CTA units/kg, these purified preparations of urokinase produce an elevation of temperature .
which varies from 0.5 to 0.9C from one rabbit to the other.
In order to obtain preparations of urokinase having still further ~ , reduced contents of pyrogenic substances recourse is had to the method according to the invention of purification, more particularly of depyrogenat-ion, from proteinic substances with biological activity, especially urokinase, which preferably has been subjected previously to a first purification, this ", method being characterised in that a partial saturation of this solution is produced with an agent for the precipitation of the proteins to a degree, which is a function of the initial concentration in the solution of the pro-teins, such that this partial saturation only induces the precipitation of a portion of the proteins contained in the initial solution, this portion ~ being sufficiently reduced for its content in the proteinic substance with ; biological ac~ivity to be maintained in predetermined low proportions, if not practically negligible, and the supernatant liquid is collected.
In a preferred embodiment of the method according to the invention, applied to urokinase, the precipitating agent is constituted by ammonium sul-phate, the above-said partial precipitation being effected on a urokinase .
solution containing from 1.5 to 3 mg of proteins per ml, at a pH comprised ~' .~: ,~ , ,;, between 3 and 4.5, preferably of the order of 3.8, the conductivity of this :~ , .. .. .
~ solution having been adjusted previously to a value comprised between 15,000 ; and 25,000 micromhos per centimetre, the degree of saturation of the solution ;`
in ammonium sulphate, comprised between 0.25 to 0.35, not being pushed beyond ,; :
that which produces precipitation of proteins containing more than 5% of the urokinase activity contained in the initial solution ~being understood that ~-the degree of saturation of a solution equals 1, when the solution is comple-tely saturated: that is to say by the solution of 700 g of ammonium sulphate in 1,000 ml of distilled water at -~4C). The urokinase practically free of pyrogenic substances contained in the supernatant liquid can then be isolated, especially by ~alting the solution with crystalline ammonium sulphate.
It will be appreciated that, taking into account the preferred ranges of concentrations of proteins indicated above, it will be advantageous to pro-ceed with a preliminary puriEication as extensive as possible of the urokinases to be treated, to eliminate the maximum possible of proteins, so that it is not necessary to operate with too large volumes of solution. In other words, the method according to the invention constitutes, in one of its preferred applica-tions, a final purification step intended to perfect the effects of a prelimina-ry purification method. ~`
Advantageously recourse will be had to the method of purification cles~;ribed above, comprising the two above-indicated exclusion chromatography steps, completed by a third step comprising:
either an absorption chromatography on carboxymethyl-cellulose of the urokinase contained in the solution resulting from combination of the effluent and the washings from the resin of the second exclusion chromatography, at a pH of the order 5, and the elution of the urokinase absorbed on carboxymethyl-cellulose with a phosphate buffer at pH 6.8 - 6.9, or an absorption of the solution resulting from the conibination of the effluent and the washings of the second exclusion chromatography on kaolin at pH 6.2, the elution of the urokinase held on the kaolin by a solution of ;1`';. ' '.,: '.
. ~L04~562 ammonium chloride in 4% ammonia, precipitation with ammonium sulphate, taking ,: -; up the precipitate in water an-l dialysis of the suspension against an 0.05 M
phosphate solution buffered to p~ 6.2, filtration of the dialysate on a column of resin of the Amberlite IRC 50 type, and elution of the resin to recover the urokinase.
It has been noted, as already indicated above, that the degree of saturation of the solution in the ammonium sulphate wnich produces the forma~
tion of a precipitated protein, (among which are the pyrogenic substances) retaining a given minimum percentage of urokinase varies as a function, espe-cially inversely, of the initial content of prokeins of the treated solution.
The values which are suitable to be given to this degree of saturation, taking into account the initial content of the solution in proteins, will be .apparant especially from the graph of the single Figure of the accompanying drawings. These values have been cletermined experimentally, especially on solut:ions purified according to the preferred method of purification indicated below, in particular on those obtained in the Examples described below rhe axis A of the single figure is graduated in concentrations of proteins (in mg/ml) of solutions to be treated, the C axis in degrees of saturation (S) of the solution of ammonium sulphate and the B axis in precent of urokinase en- '!
trained in the precipitated proteins formed under the conditions of the inven-tion. The relative arrangements of these axes and the scale of thier gradua-tions have been selected so that the percentage of urokinase entrained is, in ; each case, given by the point of intersection with the B axis of a line passing through the points representing the concentration in proteins, on the A axis, and of the degree of saturation in am~nium sulphate on the C axis respectively. .:
Thus there is obtained, for example, a protein precipitate holding 5% of the urokinase activity of the initial solution, by bringing to 0.30 the degree of saturation in ammonium sulphate of the solution, when the initial content in proteins of the latter is 2 mg/ml (dashed line a), or by bringing it to 0.25 when this initial content is 3 mg/ml ~mixed line _). `:
.. , . . , : :,. ., , . . . .. ~ . :
lO~S62 Considering for example3 the case of an initial solution of urokinase containing 2mg/ml of proteins, in more specific manner, the following observa-tions may be made.
Up to a value of the degree of saturation equal to 0.20, there is no formation of precipitate. The solution obtained hence conserves its pyrogenic substances. At the same concentration of proteins and with a degree of satura-tion equal to 0.25, a slight precipitate begins to be formed, this precipitate retaining about 2.5% of the urokinase contained in the medium. This saturation is not sufficient to retain the whole of the pyrogenic substances. On the other hand, with a degree of saturation of 0.30, the precipitate formed which contains about 5% of urokinase, retains also the larger part of the pyrogenic substances. The supernatant liquid, which still contains 95% of the total urokinase activity contained in the initial solution, is free of pyrogenic sub-stances according to the standards fixed by the French Pharmacopea already men-tioned, at doses as high as 15,000 CTA units/kg. At a saturation above 0.30, the supernatant liquid will also be a pyrogenic at the same doses but the amount of urokinase retained on the precipitate will be greater, as shown in the graph.
Additional characteristics of the invention will become apparent also in the course of the description of the ~xamples which follow. It is under-stood that in the following, all the operations are carried out at a tempera-ture of the order of 4C, except in the case where a different temperature is expressly stipulated.
Fxample I - Preparation of purified urokinase One thousand litres of urokinase are collected on phenol. The amount of phenol utilised for this purpose is such that at the end of the collection, the concentration of phenol is at a minimum 0.5%. By the addition of acetic acid, the p~ of the urine thus collected is adjusted to 5.8. ~-
2 - Absorption of the urokinase contained in the urine on hyflosupercel ;
To the mixture is added 5 kg of hyflosupercel and, in a clouble walled `;' .
_g_ ':
., ~ .
. . . : ' . . , , . . : . . ~ , ' ' : ' !
104~S62 Grignard vessel cooled to 4C, it is subjected to agitation for 3 hours. The hyflosupercel powder is collected by filtration on a filter press and 17 kg of a paste contained in 12 kg of water is obtained. ; `
,,: .,
To the mixture is added 5 kg of hyflosupercel and, in a clouble walled `;' .
_g_ ':
., ~ .
. . . : ' . . , , . . : . . ~ , ' ' : ' !
104~S62 Grignard vessel cooled to 4C, it is subjected to agitation for 3 hours. The hyflosupercel powder is collected by filtration on a filter press and 17 kg of a paste contained in 12 kg of water is obtained. ; `
,,: .,
3 - Extraction of the urokinase absorbed on tne hyflosupercel `
To the pas~e obtained is added 960 ~ of crystalline sodium chloride and 17 litres of a solution buffered to pH 7.2 obtained by adding into 1,000 ml of demineralised water, 10.8 g of disodium phosphate and 80 g of NaCl. The .. ,~,.. . .
resistivity of the buffer solution is 84,000 microhms per centimeter at 15C. : ~
After 30 minutes shaking, the hyflosupercel powder is separated from ;`~ -the mixture by filtration on a 200 mm diameter Buchner funnel, lined with a `` ~
cloth for example of synthetic fibre, such as that marketed under the trade- '`! '' ''~' mark "Tergal". The powder is allowed to deposit and the filtration is termina-ted by operating under a suction of 500 mm of mercury. Before the powder is completely dry, it is washed with 10 litres of the washing liquid, there is obtained a volume of 30 litres. The urokinase activity of the solution thus obtained is 4 million CTA units.
To the pas~e obtained is added 960 ~ of crystalline sodium chloride and 17 litres of a solution buffered to pH 7.2 obtained by adding into 1,000 ml of demineralised water, 10.8 g of disodium phosphate and 80 g of NaCl. The .. ,~,.. . .
resistivity of the buffer solution is 84,000 microhms per centimeter at 15C. : ~
After 30 minutes shaking, the hyflosupercel powder is separated from ;`~ -the mixture by filtration on a 200 mm diameter Buchner funnel, lined with a `` ~
cloth for example of synthetic fibre, such as that marketed under the trade- '`! '' ''~' mark "Tergal". The powder is allowed to deposit and the filtration is termina-ted by operating under a suction of 500 mm of mercury. Before the powder is completely dry, it is washed with 10 litres of the washing liquid, there is obtained a volume of 30 litres. The urokinase activity of the solution thus obtained is 4 million CTA units.
4 - Precipitation of the urokinase contained in the_uffer solution -. . .
The pH of the solution is adjusted to 4.2 by the addition of 5 N
hydrochloric acid. 13.5 kg of ammonium sulphate added (450 g per litre of solution) and it is agitated until complete solution of the salt. -~ ~ .
The mixture is allowed to stand for 12 hours. The precipitate which `
is formed is collected by centrifugation. For this purpose there is used a commercial centrifuge marketed under the name Alfa Laval of type B 1424 F.
There are obtained 60 g of moist precipitate, there is added to the precipi-.,i tate 60 ml of an aqueous solution containing 18 g of glucose per litre of water.
The mixture is then dialysed until thP final conductivity of the so-lution containing the urokinase is 22,000 micromhos.
It is activated by shaking. The dialysed solution is clarified by centrifugation. The activity of the solution obtained is 4~000~000 CTA units . ~' ~ . ' ' ,':; ':
~04~56Z :
of urokinase.
The pH of the solution is adjusted to 4.2 by the addition of 5 N
hydrochloric acid. 13.5 kg of ammonium sulphate added (450 g per litre of solution) and it is agitated until complete solution of the salt. -~ ~ .
The mixture is allowed to stand for 12 hours. The precipitate which `
is formed is collected by centrifugation. For this purpose there is used a commercial centrifuge marketed under the name Alfa Laval of type B 1424 F.
There are obtained 60 g of moist precipitate, there is added to the precipi-.,i tate 60 ml of an aqueous solution containing 18 g of glucose per litre of water.
The mixture is then dialysed until thP final conductivity of the so-lution containing the urokinase is 22,000 micromhos.
It is activated by shaking. The dialysed solution is clarified by centrifugation. The activity of the solution obtained is 4~000~000 CTA units . ~' ~ . ' ' ,':; ':
~04~56Z :
of urokinase.
5 - Exclusion of the urokinase n DEAE cellulose a) 7.1 g of DEAE cellulose is added to the preceding clarified solu-tion and the pll is brought to 4.5. After 30 minutes of agitation, the DEAE
cellulose is separated by filtration on a lS0 mm diameter Buchner funnel by operating under a moderate vacuum to prevent the formation of froth.
The DEAB cellulose is washed in two stages with a solution of ammo-nlum sulphate at a conductivity of 15,000 micromhos per centimetre, at pH 4.5.
The volume used for each washing is equal to that of the DEAE cellulose and represents about 250 ml.
During the filtration of the washing solution the flask is kept under vacuum in an ice bath.
The activity of the filtrate is 4,000,U00 C'l'A units.
b) To the filtrate obtained, pH 4.5, is added 17.2 g of DEAE cellu~
lose brought to pH 6.7.
The conductivity i5 lowered by dilution with water to 9,000 micromhos per centimetre, After 30 minutes of agitation, the DEAE cellulose is separa-ted by filtration on a porcelain Buchner funnel of 150 mm diameter. Progres-sively with the filtration, the pH of the filtrates or effluents collected is brought to 5 by the addition of S N sulfuric acid.
The DBAE cellulose is washed with a solution of ammonium sulphate, of 9,000 micromhos per centimetre conductivity, at pH 7, until a colourless . .
filtrate is obtained.
The filtrates and washing solutions are combined and adjusted to pH
5. The urokinase activity of the solution obtained is 4,400,000 CTA units. ;
cellulose is separated by filtration on a lS0 mm diameter Buchner funnel by operating under a moderate vacuum to prevent the formation of froth.
The DEAB cellulose is washed in two stages with a solution of ammo-nlum sulphate at a conductivity of 15,000 micromhos per centimetre, at pH 4.5.
The volume used for each washing is equal to that of the DEAE cellulose and represents about 250 ml.
During the filtration of the washing solution the flask is kept under vacuum in an ice bath.
The activity of the filtrate is 4,000,U00 C'l'A units.
b) To the filtrate obtained, pH 4.5, is added 17.2 g of DEAE cellu~
lose brought to pH 6.7.
The conductivity i5 lowered by dilution with water to 9,000 micromhos per centimetre, After 30 minutes of agitation, the DEAE cellulose is separa-ted by filtration on a porcelain Buchner funnel of 150 mm diameter. Progres-sively with the filtration, the pH of the filtrates or effluents collected is brought to 5 by the addition of S N sulfuric acid.
The DBAE cellulose is washed with a solution of ammonium sulphate, of 9,000 micromhos per centimetre conductivity, at pH 7, until a colourless . .
filtrate is obtained.
The filtrates and washing solutions are combined and adjusted to pH
5. The urokinase activity of the solution obtained is 4,400,000 CTA units. ;
6 - Absorption of the urokinase on kaolin and separation by elution. , ;
Kaolin is prepared as follows:
1 kg of kaolin is washed and placed in suspension in 3 litres of dis-; tilled water. The mixture is left for 90 minutes at 120C in the autoclave.
After having allowed the material in suspension to settle, the kaolin is separated then placed to dry in the oven at 200C.
, .
-11- . ' ~.
':.:
:.' .:: ~' ' S6~ ` ~
The solution obtained in the course of the last step titrating 4,400,000 CTA units is brought to a pH of 6.8, then after the addition of 65 g of kaolin prepared in the above-indicated manner, to a pH of 6.2. The mixture is subjected to agitation for one hour. The kaolin is then separated by cen-trifugation in the cold and placed in suspension in a litre of 0.05 M phos- `
phate solution buffered to pH 6.2. , After 30 minutes of shaking, the kaolin is separated by cent ~ fuga-tion. `!,~
; The 65 g of kaolin are placed in suspension in 500 ml of a solution ;
containing 75 g of ammonium chloride per litre of 4% ammonia, and agitated for 30 minutes. The kaolin, separated from the supernatant liquid by centrifuga- 2~ -:-~ tion, is subjected to the same elution operation twice additionally.
; The supernatant liquids obtained Oll the centri~ugations are com-j bined and 0,1 N sulphuric acid added to obtain a pH of 3.5.
~ y combining the supernatant liquids obtained on the centrifugation, 1,400 ml of eluate are obtained. The pH of the eluate is brought to 3.5 by the addition of 0.1 N sulphuric acid. There is then added 490 g of ammonium `
I sulphate ~350 g per litre of eluate). The precipitate formed by centrifuga-; tion in the cold is collected and it is placed in suspension in water. There s, 20 is then carried out for 18 hours a dialysis against a 0.05 M phosphate solu- `~
tion buffered to pH 6.2.
The urokinase activity of the solution obtained is 2,800,000 CTA
units.
Kaolin is prepared as follows:
1 kg of kaolin is washed and placed in suspension in 3 litres of dis-; tilled water. The mixture is left for 90 minutes at 120C in the autoclave.
After having allowed the material in suspension to settle, the kaolin is separated then placed to dry in the oven at 200C.
, .
-11- . ' ~.
':.:
:.' .:: ~' ' S6~ ` ~
The solution obtained in the course of the last step titrating 4,400,000 CTA units is brought to a pH of 6.8, then after the addition of 65 g of kaolin prepared in the above-indicated manner, to a pH of 6.2. The mixture is subjected to agitation for one hour. The kaolin is then separated by cen-trifugation in the cold and placed in suspension in a litre of 0.05 M phos- `
phate solution buffered to pH 6.2. , After 30 minutes of shaking, the kaolin is separated by cent ~ fuga-tion. `!,~
; The 65 g of kaolin are placed in suspension in 500 ml of a solution ;
containing 75 g of ammonium chloride per litre of 4% ammonia, and agitated for 30 minutes. The kaolin, separated from the supernatant liquid by centrifuga- 2~ -:-~ tion, is subjected to the same elution operation twice additionally.
; The supernatant liquids obtained Oll the centri~ugations are com-j bined and 0,1 N sulphuric acid added to obtain a pH of 3.5.
~ y combining the supernatant liquids obtained on the centrifugation, 1,400 ml of eluate are obtained. The pH of the eluate is brought to 3.5 by the addition of 0.1 N sulphuric acid. There is then added 490 g of ammonium `
I sulphate ~350 g per litre of eluate). The precipitate formed by centrifuga-; tion in the cold is collected and it is placed in suspension in water. There s, 20 is then carried out for 18 hours a dialysis against a 0.05 M phosphate solu- `~
tion buffered to pH 6.2.
The urokinase activity of the solution obtained is 2,800,000 CTA
units.
7 - Filtration of the dialysate on a column of resin ;
..
The dialysed solution is filtered on a column of resin of the Amberlite IRC 5Q type 4cm in diameter and 60 cm in height, previously equi-i librated with 0.05 M phosphate solution buffered to pH 6.6.
When the eluate is run from the column, the resin is washed with the buffer solution until the addition of 5% trichloracetic acid no longer ' 30 causes the formation of a precipitate in the fractions collected.
To elute the urokinase, there is used a buffer solution of 0.2 M
phosphate, of pH 8.2, containing 75 g of ammonium chloride per litre of solu-, ''' '.
~L(14(~56Z
.
tion. The eluate is collected in a fraction collector. The tubes containing the activity are precipitated by the ammonium sulphate at pH 3.5 to precipi-tate the urokinase present, in the proportion of ~50 g of ammonium sulphate per litre of elluate, namely 15,'3 g for the 450 ml of eluate obtained.
Example II - Pre~aration of purified urokinase , - A precipitate in ammonium sulphate containing crude urokinase and obtained under the conditions indicated in paragraph 4 of example I , is treated as follows, starting with a solution itself obtained from human urine, to which the prior concentration method according to paragraphs 1 to 3 of the sams example I , has been applied.
A) Conditions of the first exclusion : .
The precipitate in ammonium sulphate is solubilised in one to two parts of glucose water, in the proportion oE 18 grammes of glucose per litre and the pH is adjusted to a value comprised between 4 and 6 and preferably equal to 4.5.
: The solution obtained is dialysed against distilled water until there is obtained a conductivity comprised between 30,000 micromhos per cen-timetre and 50~000 micromhos per centimetre, preferably equal to 40,000 microm-hos per centimetre.
l 20 The solution obtained is centrifuged to 3,000 g to eliminate the in-!' solubles formed constituted by the non-proteinic impurities.
A further dialysis oE the supernatant liquid is carried out until a ~ `dialysate is obtained whose conductivity is comprised between 15,000 and ... ,. ~ .
25,000 micromhos per centimetre, preferably equal to 22,000 micromhos per cen-timetre.
D~AE cellulose is added to the solution obtained in the proportion of ,::
25 g of dry resin or 100 g af drained resin previously equilibrated with an ~` 0.11 M ammonium sulphate solution at pH 4.5 (conductivity of 15,000 micromhos per centimetre at 4C), by 14 million CTA units of urokinase.
The medium is shaken for a duration of 20 to 40 minutes, preferably :
~04~56;: ~
30 minutes.
The resin is drained, especially on a Buchner under slight suction and .
it is washed with a solution of 0.11 M ammonium sulphate at pH 4.5 until a colourless effluent or filtrate is obtained.
The filtrate and the washing liquids are combined. The total uroki-nase activity of the solution obtained is substantially equal to that which was used at the start.
B) Conditions of the second exclusion The above solution is brought to a pH comprised between 6 and 7, pre~
ferably of the order of 6.6. ;~
; To this solution 60 g of DEAE cellulose powder or 250 g of the drained DEAE cellulose is added, previoulsy equilibrated with an 0.068 M ammonium sulphate solution to pH 7 (conductivity 9,000 micromhos per centimetre at 4C), by 14 million CTA units of urokinase. I'he conductivity of the medium is then 15,000 to 18,000 micromhos per centimetre. `
'rhe conductivity of the medium is then brought to 9,000 micromhos per centimetre by successive additions of apyrogenic distilled water, so that each addition produces a drop in conduct.vity of the medium of 1,000 micromhos per centimetre each addition being separated by an agitation for a duration -of 5 to 15 minutes, preferably equal to 10 minutes.
The pH of the medium is then adjusted to 7 by the addition of 2 M
soda. After agitation for a duration of 30 minutes, it is filtered, especially ~ ;
on the stainless Buchner under slight suction, the receiver vessel being kept in a ~elting ice bath, the resin is washed with a solution of ammonium sulphate of 9JOOO micromhos conductivity, 0.068 M and the pH 7 until a colourless fil-trate is obtained.
The above said solution~ brought to pH 5J is treated with carboxy- `
methylcellulose in the proportion of 80 g of dry resin per 14JOOO rA units of urokinase, the carboxymethylcellulose having previously been equilibrated with a solution of ammonium sulphate at 9,000 ~Mhos per centimeterJ pH 5~ = 0.068 M.
~ ' -14- ~`
. .
~ ' ~
:
~04056Z `
The suspension is kept moderately shaken for a duration of 12 to 14 hours at a - temperature comprised between 2 and 4C, its pH being kept at 5 - 0.1.
The suspension is then filtered on crimped paper through a porcelain Buchner funnel under slight suction. The ef1uents conserve the initial colour.
The carboxymethylcellulose is then washed with a solution of ammonium sulphate of 9,000 micromhos per centimetre at pH 5, then by a phosphate buffer 0.05 M at pH 6.5, for the pu~pose of removing the sulphates, until a colour-less solution is obtained.
The carboxymethylcellulose is then drained. There is then added to the latter a volume of 0.5 M phosphate buffer equal to the weight of the 0.05 M buffer retained in the drained carboxymethylcellulose at a temperature of 15 - 20C with shaking. There is next added an equal volume of an 0.29 M
sodium phosphate buffer, then an 0.5 M phosphate buffer, to obtain a final ~.
molarity of 0.29 M at a p~l of the order of 6.8 to 6.9. ;;
The temperature is then brought to 6C, the suspension is shaken for ,! ' . .:
30 minutes, the carboxymethylcellulose being then drained on crimped paper -~
(on the Buchner funnel) and washed with an 0.29 M phosphate buf~er until a - ~ -clear solution is obtained which does not precipitate with tannin.
I'he elluate and the washing liquids are combined to form a purified -` ;
solution of urokinase containing 80 to 90% of the urokinase activi.ty of the solution resulting from the combination of the effluent and of the washing ," ~. .
solutions obtained at the end of the second above said exclusion chromato-graphy.
~xample III - Preparation of apyrogenic urokinase lhe final precipitate of example I is collected by centrifugation, and dissolved in apyrogenic and steri~ distilled water~ so as to obtain the conductivity comprised between 15,000 and 20,000 micromhos per centimetre, The concentration of this solution of proteins is of the order of 2 mg/ml.
The p~ being adjusted to the value of 3.8 and the temperature com-30 prised between 0 ~nd 4C, there is added to this medium a saturated solution : ;
.". '`'`~ '',;.
~, ~, ' .' '"' ' .
:::
104~S6;~
of ammonium sulphate until a saturation of 0.3 is obtained (namely a solution of 1.22 M ammonium sulphate). The medium is shaken for 15 minutes, then left to stand, also for 15 minutes.
The precipitate is collected by contrifugation at 10,000 g (fraction A)-. "
To the supernatent liquid is added pure ammonium sulphate, in a pro-portion of 250 g/l, at ptl 3-3 5, then shaken for 30 minutes. The precipitate . containing the urokinase is collected by centrifugation at 10,000 g (fraction - ~
~ B) e .
9 - Results ~ach of these precipitates (fractions A and B) is restored to suspen-sion in apyrogenic and sterile distilled water, the ptl of the suspension being brought to 7J the suspension then being finally filtered on a 0.22 ~ membrane in a sterile medium.
The filtrate coming from fraction A contains about 5% of urokinase ;
activity.
The filtrate coming from fraction B contains about 95% of urokinase ~
activity of the initial solution. ;
The fraction A shows itself in practice to retain the whole of the ;
pyrogenic substances initially contained in the initial solution obtained by dissolving the precipitate in ammonium sulphate before fractionation. The fraction B is on the other hand free of pyrogenic substances according to French Pharmacopea standard;, loc-cit. These results will be evident from table 1 below in which there are reported the increases in temperature obser-ved respectively in separate series of three rabbits for each of the fractions tested, and in an additional series of eight rabbits for fraction B, all these rabbits having received the doses indicated in the left hand portion of the table, under the conditions provided by the French Pharmacopea.
.1 . .
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h o o o ...... _ .,`,;'`'~' '','., :'. ~' ' ' '.............................................. .~
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h a~ O ¢ ¢ ¢ ~ ¢
O O U~ ~ O ~ O ' .,~ ~J ~1 .,~ o ......
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~ 41 ~R h ~ ~ '....... .
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the addition of several drops of 2 N sulphuric acid. The precipitate formed is collected by centrifugation at 10,000 g and the supernatent liquid is sepa- ;~
`; rated: there are obtained 25 g of precipitate ~fraction D). , C) Results The precipitate of fraction C is dissolved in 25 ml of sterile apyro-; genic distilled water, and the pH is brought to 7 by 0.1 N soda. The solu-tion obtained is filtered under sterile conditions: .
. its titre in urokinase is equal to 10,000 CTA units/ml. The precipi-tate of fraction D is dissolved in 150 ml of sterile apyrogenic distilled ~ 10 water. The pH is brought to 7 by 0.1 N soda and the solution obtained is ,; filtered under sterile conditions.
. ~. :, f Its titre in urokinase is equal to 300,000 CTA units/ml.
Its specific activity to protein is ~3,000 CTA units per mg of pro-teins.
The same observations can be drawn as in the previous case. It is . ,j .
observed that the major portion of the pyrogenic subStances has been retained in fraction C, whilst fraction D is free o pyrogenic substances according to the French Pharmacopea standards, as will be apparent from ta le II below ~ -established under similar conditions to those relating with respect to table : 20 It is self-evident that instead of ammonium sulphate there may be used any other salt known for its aptitude to precipitate proteins, such as, for example, magnesium sulphate, sodium chloride, etc., it being underskood , that it is necessary in each case, to determine previously the degree of :~ saturation of the initial solution of the salt concerned whlch would lead to -,~i a precipitation of proteins under the above-indicated conditions. : -:i ,' ,',.'1 ;,' :~ -18-, ii :
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h rl) ~ ~ ~ . . .
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. . .
.. .. .
~gJ4~)562 i . As a result of which there is obtained a method of depyrogenation, .
and also of concentration relative to the urokinase with regard to its pro- :;
~ tein content, which is simple and particularly effective, with a yield of at .. . .
least 95% of urokinase activity, the urokinase obtained, apyrogenic at high doses, being then administrable to patients without any secondary effects at .. . .
doses of 150,000 uCTA or more, in a single in~ection by the intravenous route, . -for the treatment of the above-mentioned disorders, either as is, or in .. .
another form, for example after conversion into urokinase heparinate, for ~: :example under the conditions described in our copending application serial number 152,328.
~ , .
. ~`' In this disclosure and claims reference is made to the enzyme -r . ~
urokinase. According to the scheme of M. Florkin and E. H. Stotz, published in "Comprehenslve Biochemistry", Vol. 13 (3rd Edition, Elsevier Publishing Co., New York, lg73) this enzyme has the classification number 3 4.99 26.
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The dialysed solution is filtered on a column of resin of the Amberlite IRC 5Q type 4cm in diameter and 60 cm in height, previously equi-i librated with 0.05 M phosphate solution buffered to pH 6.6.
When the eluate is run from the column, the resin is washed with the buffer solution until the addition of 5% trichloracetic acid no longer ' 30 causes the formation of a precipitate in the fractions collected.
To elute the urokinase, there is used a buffer solution of 0.2 M
phosphate, of pH 8.2, containing 75 g of ammonium chloride per litre of solu-, ''' '.
~L(14(~56Z
.
tion. The eluate is collected in a fraction collector. The tubes containing the activity are precipitated by the ammonium sulphate at pH 3.5 to precipi-tate the urokinase present, in the proportion of ~50 g of ammonium sulphate per litre of elluate, namely 15,'3 g for the 450 ml of eluate obtained.
Example II - Pre~aration of purified urokinase , - A precipitate in ammonium sulphate containing crude urokinase and obtained under the conditions indicated in paragraph 4 of example I , is treated as follows, starting with a solution itself obtained from human urine, to which the prior concentration method according to paragraphs 1 to 3 of the sams example I , has been applied.
A) Conditions of the first exclusion : .
The precipitate in ammonium sulphate is solubilised in one to two parts of glucose water, in the proportion oE 18 grammes of glucose per litre and the pH is adjusted to a value comprised between 4 and 6 and preferably equal to 4.5.
: The solution obtained is dialysed against distilled water until there is obtained a conductivity comprised between 30,000 micromhos per cen-timetre and 50~000 micromhos per centimetre, preferably equal to 40,000 microm-hos per centimetre.
l 20 The solution obtained is centrifuged to 3,000 g to eliminate the in-!' solubles formed constituted by the non-proteinic impurities.
A further dialysis oE the supernatant liquid is carried out until a ~ `dialysate is obtained whose conductivity is comprised between 15,000 and ... ,. ~ .
25,000 micromhos per centimetre, preferably equal to 22,000 micromhos per cen-timetre.
D~AE cellulose is added to the solution obtained in the proportion of ,::
25 g of dry resin or 100 g af drained resin previously equilibrated with an ~` 0.11 M ammonium sulphate solution at pH 4.5 (conductivity of 15,000 micromhos per centimetre at 4C), by 14 million CTA units of urokinase.
The medium is shaken for a duration of 20 to 40 minutes, preferably :
~04~56;: ~
30 minutes.
The resin is drained, especially on a Buchner under slight suction and .
it is washed with a solution of 0.11 M ammonium sulphate at pH 4.5 until a colourless effluent or filtrate is obtained.
The filtrate and the washing liquids are combined. The total uroki-nase activity of the solution obtained is substantially equal to that which was used at the start.
B) Conditions of the second exclusion The above solution is brought to a pH comprised between 6 and 7, pre~
ferably of the order of 6.6. ;~
; To this solution 60 g of DEAE cellulose powder or 250 g of the drained DEAE cellulose is added, previoulsy equilibrated with an 0.068 M ammonium sulphate solution to pH 7 (conductivity 9,000 micromhos per centimetre at 4C), by 14 million CTA units of urokinase. I'he conductivity of the medium is then 15,000 to 18,000 micromhos per centimetre. `
'rhe conductivity of the medium is then brought to 9,000 micromhos per centimetre by successive additions of apyrogenic distilled water, so that each addition produces a drop in conduct.vity of the medium of 1,000 micromhos per centimetre each addition being separated by an agitation for a duration -of 5 to 15 minutes, preferably equal to 10 minutes.
The pH of the medium is then adjusted to 7 by the addition of 2 M
soda. After agitation for a duration of 30 minutes, it is filtered, especially ~ ;
on the stainless Buchner under slight suction, the receiver vessel being kept in a ~elting ice bath, the resin is washed with a solution of ammonium sulphate of 9JOOO micromhos conductivity, 0.068 M and the pH 7 until a colourless fil-trate is obtained.
The above said solution~ brought to pH 5J is treated with carboxy- `
methylcellulose in the proportion of 80 g of dry resin per 14JOOO rA units of urokinase, the carboxymethylcellulose having previously been equilibrated with a solution of ammonium sulphate at 9,000 ~Mhos per centimeterJ pH 5~ = 0.068 M.
~ ' -14- ~`
. .
~ ' ~
:
~04056Z `
The suspension is kept moderately shaken for a duration of 12 to 14 hours at a - temperature comprised between 2 and 4C, its pH being kept at 5 - 0.1.
The suspension is then filtered on crimped paper through a porcelain Buchner funnel under slight suction. The ef1uents conserve the initial colour.
The carboxymethylcellulose is then washed with a solution of ammonium sulphate of 9,000 micromhos per centimetre at pH 5, then by a phosphate buffer 0.05 M at pH 6.5, for the pu~pose of removing the sulphates, until a colour-less solution is obtained.
The carboxymethylcellulose is then drained. There is then added to the latter a volume of 0.5 M phosphate buffer equal to the weight of the 0.05 M buffer retained in the drained carboxymethylcellulose at a temperature of 15 - 20C with shaking. There is next added an equal volume of an 0.29 M
sodium phosphate buffer, then an 0.5 M phosphate buffer, to obtain a final ~.
molarity of 0.29 M at a p~l of the order of 6.8 to 6.9. ;;
The temperature is then brought to 6C, the suspension is shaken for ,! ' . .:
30 minutes, the carboxymethylcellulose being then drained on crimped paper -~
(on the Buchner funnel) and washed with an 0.29 M phosphate buf~er until a - ~ -clear solution is obtained which does not precipitate with tannin.
I'he elluate and the washing liquids are combined to form a purified -` ;
solution of urokinase containing 80 to 90% of the urokinase activi.ty of the solution resulting from the combination of the effluent and of the washing ," ~. .
solutions obtained at the end of the second above said exclusion chromato-graphy.
~xample III - Preparation of apyrogenic urokinase lhe final precipitate of example I is collected by centrifugation, and dissolved in apyrogenic and steri~ distilled water~ so as to obtain the conductivity comprised between 15,000 and 20,000 micromhos per centimetre, The concentration of this solution of proteins is of the order of 2 mg/ml.
The p~ being adjusted to the value of 3.8 and the temperature com-30 prised between 0 ~nd 4C, there is added to this medium a saturated solution : ;
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~, ~, ' .' '"' ' .
:::
104~S6;~
of ammonium sulphate until a saturation of 0.3 is obtained (namely a solution of 1.22 M ammonium sulphate). The medium is shaken for 15 minutes, then left to stand, also for 15 minutes.
The precipitate is collected by contrifugation at 10,000 g (fraction A)-. "
To the supernatent liquid is added pure ammonium sulphate, in a pro-portion of 250 g/l, at ptl 3-3 5, then shaken for 30 minutes. The precipitate . containing the urokinase is collected by centrifugation at 10,000 g (fraction - ~
~ B) e .
9 - Results ~ach of these precipitates (fractions A and B) is restored to suspen-sion in apyrogenic and sterile distilled water, the ptl of the suspension being brought to 7J the suspension then being finally filtered on a 0.22 ~ membrane in a sterile medium.
The filtrate coming from fraction A contains about 5% of urokinase ;
activity.
The filtrate coming from fraction B contains about 95% of urokinase ~
activity of the initial solution. ;
The fraction A shows itself in practice to retain the whole of the ;
pyrogenic substances initially contained in the initial solution obtained by dissolving the precipitate in ammonium sulphate before fractionation. The fraction B is on the other hand free of pyrogenic substances according to French Pharmacopea standard;, loc-cit. These results will be evident from table 1 below in which there are reported the increases in temperature obser-ved respectively in separate series of three rabbits for each of the fractions tested, and in an additional series of eight rabbits for fraction B, all these rabbits having received the doses indicated in the left hand portion of the table, under the conditions provided by the French Pharmacopea.
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h o o o ...... _ .,`,;'`'~' '','., :'. ~' ' ' '.............................................. .~
~ R ~4 .
h a~ O ¢ ¢ ¢ ~ ¢
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~ 41 ~R h ~ ~ '....... .
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the addition of several drops of 2 N sulphuric acid. The precipitate formed is collected by centrifugation at 10,000 g and the supernatent liquid is sepa- ;~
`; rated: there are obtained 25 g of precipitate ~fraction D). , C) Results The precipitate of fraction C is dissolved in 25 ml of sterile apyro-; genic distilled water, and the pH is brought to 7 by 0.1 N soda. The solu-tion obtained is filtered under sterile conditions: .
. its titre in urokinase is equal to 10,000 CTA units/ml. The precipi-tate of fraction D is dissolved in 150 ml of sterile apyrogenic distilled ~ 10 water. The pH is brought to 7 by 0.1 N soda and the solution obtained is ,; filtered under sterile conditions.
. ~. :, f Its titre in urokinase is equal to 300,000 CTA units/ml.
Its specific activity to protein is ~3,000 CTA units per mg of pro-teins.
The same observations can be drawn as in the previous case. It is . ,j .
observed that the major portion of the pyrogenic subStances has been retained in fraction C, whilst fraction D is free o pyrogenic substances according to the French Pharmacopea standards, as will be apparent from ta le II below ~ -established under similar conditions to those relating with respect to table : 20 It is self-evident that instead of ammonium sulphate there may be used any other salt known for its aptitude to precipitate proteins, such as, for example, magnesium sulphate, sodium chloride, etc., it being underskood , that it is necessary in each case, to determine previously the degree of :~ saturation of the initial solution of the salt concerned whlch would lead to -,~i a precipitation of proteins under the above-indicated conditions. : -:i ,' ,',.'1 ;,' :~ -18-, ii :
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. . .
.. .. .
~gJ4~)562 i . As a result of which there is obtained a method of depyrogenation, .
and also of concentration relative to the urokinase with regard to its pro- :;
~ tein content, which is simple and particularly effective, with a yield of at .. . .
least 95% of urokinase activity, the urokinase obtained, apyrogenic at high doses, being then administrable to patients without any secondary effects at .. . .
doses of 150,000 uCTA or more, in a single in~ection by the intravenous route, . -for the treatment of the above-mentioned disorders, either as is, or in .. .
another form, for example after conversion into urokinase heparinate, for ~: :example under the conditions described in our copending application serial number 152,328.
~ , .
. ~`' In this disclosure and claims reference is made to the enzyme -r . ~
urokinase. According to the scheme of M. Florkin and E. H. Stotz, published in "Comprehenslve Biochemistry", Vol. 13 (3rd Edition, Elsevier Publishing Co., New York, lg73) this enzyme has the classification number 3 4.99 26.
~ . .
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Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE D AS FOLLOWS:
1. A method of purifying a urokinase solution mixed with foreign proteins and pyrogenic substances, which comprises partially saturating the solution with ammonium sulphate up to a degree which causes a minor proportion, not exceeding 5%, of the total activity of the urokinase in the initial sol-ution to be precipitated with part of the proteins initially contained in said solution and collecting the supernatant liquid containing the urokinase sub-stantially freed from pyrogenic substances.
2. A method of purification according to claim 1 in which the protein concentration of the urokinase-containing solution to be purified is between 1.5 and 3 mg per ml, and in which the pH of the solution is adjusted to between 3 and 4.5 and the conductivity thereof is adjusted to between 15,000 and 25,000 micromhos/cm, the concentration of ammonium sulphate required (allowing for the initial protein concentration of the solution) for forming a precipitate containing 2-5% of the urokinase activity in the solution is de-termined, the quantity of ammonium sulphate required to obtain the thus determined concentration is added to the solution, and the supernatant liquid is collected.
3. A method according to claim 2, in which the ammonium sulphate con-centration of the solution is adjusted to a value corresponding to a degree of saturation of the solution equal to 0.3.
4. A method according to claim 1 in which the initial urokinase is obtained from human urine by: (a) processing human urine with a filtration adjuvant; (b) re-extracting the urokinase and adsorbed proteins from the fil-tration adjuvant; (c) salting out the urokinase from the solution used for extraction, by means of ammonium sulphate; (d) dissolving the resulting pre-cipitate in a glucose solution and dialysing the latter to obtain a concentrated crude-urokinase solution having a conductivity between 15,000 and 25,000 micromhos/cm, preferably approximately 22,000 micromhos/cm; (e) performing a first exclusion chromatography operation on the solution at a pH between 4 and 6 by contacting the solution with a DEAE cellulose resin; (f) performing a second exclusion chromatography operation on the solution obtained by com-bining the effluent and the washing liquors for the resin used in the first chromatography operation after adjusting the pH, if necessary, to between 6 and 7 and adjusting the conductivity of the solution to at least 15,000 micromhos/cm, the second chromatography operation being performed on DEAE
cellulose resin used in an amount from 30-60 g per 10 million CTA units of urokinase activity, the conductivity of the medium being progressively brought to a value of 9,000 micromhos/cm; and (g) either absorbing the urokinase in the solution obtained by combining the effluent and the washing liquors for the resin used in the second chromatography operation by chromatography on carboxymethylcellulose at a pH of about 5 and eluting the urokinase adsorbed on the carboxymethylcellulose using a phosphate buffer at pH 6.8 to 6.9, or absorbing urokinase from the solution obtained by combining the effluent and the washing liquors in the second chromatography operation on kaolin at pH 6.2, and eluting the urokinase retained on the kaolin with a 4% solution of ammonium chloride in ammonia, followed by precipitation with ammonium sulphate and dialysis against an 0.05 M phosphate solution buffered to pH 6.2.
cellulose resin used in an amount from 30-60 g per 10 million CTA units of urokinase activity, the conductivity of the medium being progressively brought to a value of 9,000 micromhos/cm; and (g) either absorbing the urokinase in the solution obtained by combining the effluent and the washing liquors for the resin used in the second chromatography operation by chromatography on carboxymethylcellulose at a pH of about 5 and eluting the urokinase adsorbed on the carboxymethylcellulose using a phosphate buffer at pH 6.8 to 6.9, or absorbing urokinase from the solution obtained by combining the effluent and the washing liquors in the second chromatography operation on kaolin at pH 6.2, and eluting the urokinase retained on the kaolin with a 4% solution of ammonium chloride in ammonia, followed by precipitation with ammonium sulphate and dialysis against an 0.05 M phosphate solution buffered to pH 6.2.
5. A method according to claim 4 in which in step (e), from 15 to 40 g of DEAE cellulose resin is used per 14 million CTA urokinase units.
6. A method according to claim 11 in which in step (f) from 30 to 60 g of DEAE cellulose resin is used per 10 million CTA urokinase units.
7. A method according to claim 4 or claim 5 in which in step (f) the conductivity of the solution is adjusted to from 15,000 to 18,000 micromhos/
cm.
cm.
8. A method according to any one of claims 1, 2 or 4, in which the supernatant liquid is precipitated with an excess of the precipitating agent and the precipitate is separated by centrifuging.
9. A method according to any one of claims 1, 2 or 4, in which the precipitate is dissolved at pH 7 in distilled, sterile apyrogenic water.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA152,328A CA986013A (en) | 1971-09-24 | 1972-09-22 | Methods for preparing depyrogenising and stabilising urokinase and compositions obtained thereby |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1040562A true CA1040562A (en) | 1978-10-17 |
Family
ID=4094499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA240,777A Expired CA1040562A (en) | 1972-09-22 | 1975-12-01 | Methods for purifying urokinase preparations |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1040562A (en) |
-
1975
- 1975-12-01 CA CA240,777A patent/CA1040562A/en not_active Expired
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