CA1206903A - Process for the preparation of a plasminogen activator - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A process for the preparation of a plasminogen activator is described, which comprises bringing normal diploid cells derived from human bodies capable of producing the plasminogen activator into contact with a solution containing an enzymatically-decomposed animal meat peptone, the plasminogen activator having the following properties:
a) molecular weight: 63,000 ? 10,000;
b) isoelectric point: 7.0 to 8.5;
c) affinity to fibrin: present;
d) affinity to Concanavalin A: present;
e) optimum pH: 7 to 9.5; and f) no reactivity with anti-urokinase specific antibody.
The plasminogen activator can be obtained in a high yield.

Description

"` ~2~ L)3 PROCESS FOR THE PREPARATION OF
A PLASMINOGEN ACTIVATOR

FIELD OF THE INVENTION
;This invention relates to a process for the preparation of a plasminogen activator in a high yield by ~he use of normal diploid cells derived from human bodies.
BACKGROUND OF THE INVENTION
Urokinase separated and purified from urine or cultured kidney cells and streptokinase recovered from streptococci are nowadays practically use~ as Elasminogen activators. Specifically, these plasminogen activators are used as thrombolytic agents.
However, it is known that these plasminogen activators are often administered to patients in large amounts in order to obtain necessary therapeutic effects overcoming their poorness in affinity with fibrin and thus cause side effects such as internal hemorrhage.
Specifically, plasmin which is produced in circulating ~lood by these plasminogen activators tends to lose activity immediately upon combination with plasmin inhibitors in the blood. Accordingly, in order to exhibit the neces.sary therapeutic effects, these plasminogen activators must be administered in large amounts to produce Flasmin, viz,, in an amount exceeding that of the plasmin inhibitors in the blood. However, the production of a large amount of plasmin will decompose fibrinogen, resulting in hemorrhage.
Accordingly, if a plasminogen activator which has a hlgh affinity with fibrin and is capable of producing plasmin on fibrin can be obtained, it becomes possible to deco~pose fibrin with a small amount of the plasminogen activator without being subjected to the influence of plasmin inhibitors in circulating blood, and also to reduce the effects ~f decomposing fibrinogen. Therefore, it has been desired to provide a thrombolytic agent having a high affinity for f~brin which shows a high thrombolytic activity ~sing a small amount thereof, and which has less side effects.
One of the inventors, with others, has already founa a novel plasminogen activator having properties listed below in a culture liquid of normal diploid cells or human bodies and endeavored to put it in practical use (European Patent Application (OPI) No. 0100982).
a) molecular weight: 63,000 + 10,000 b) isoelectric point: 7.0 to 8.5 c) affinity to fibrin: present d) affinity to Concanavalin A: present e) optimum pH: 7.0 to 9.S
f) no reactivity with anti-urokinase specific antibody.
However, because of low productivity, this plasminogen activator has been difficult to provide in ~2069(~3 large amounts on an ~ndustrial scale.
SUL~MAP~Y OF THE INVENTION
As a result of concentrated-studies to find an efficient process for the preparation of the plasminogen activator, if has now been found that the presence of a large amount of peptone derived by the enzymatic decompo-sition of animal meats in a solution which produces the plasminogen activator upon contact with normal diploid cells (i.e., cells having a normal diploid chromosome) 1~ derived from human bodies causes a drastic increase of the production of the activator. The present invention has been achieved on the basis of this discovery.
This invention is therefore a process for the preparation of a plasminogen activator which comprises bringing normal diploid cells derived from human bodies capable of producing a plasminogen activator into contact with a solution containing an enzymatically-decomposed animal meat peptone.
BXIEF DESCRIPTIO.~ OF THE DRAWINGS
Fig. 1 is a graph showing the optimum pH of a plasminogen activator obtained by the inventlon; and Fig~ 2 is a graph showing the results of the measurement of the yield of the plasminogen activator in the culture liquid in Example 2.

12069(D3 DET~ILED DESC~IPTION OF TH~; INVE~ITION
The present invention is achieved by the use of normal diploid cells derived from human bodies capable of producing the plasminogen activator. As such normal diploid cells, there can be used, for example, cells derived from human kidney, intestines, lung, heart, ureter, skin, foreskin, tongue, thyroid gland, placenta and womb and cells derived from the whole embryo, more preferably cells derived from human lung or foreskin. The cells derived from human bodies described above herein include cells derived from fetus and neonate.
rlhese cells can be proliferated in accordance with a normal method for the culture of animal cells, for example, as described in P.K. ~ruse and M.K. Patterson, Tissue Culture Methods and Applications, pp. 220-223, Academic Press, ~ew York,San Francisco, 1973, and thereafter they can be brought into contact with a solution containing a carbon source, a nitrogen source, and, if necessary, inorganic salts and/or other additives, to produce the plasminogen activator. ~s additives which are allowed to coexist in the solution, there can be used amino acids, vitamins, peptides, saccharides, and organic acids.
Examples of such additives include the natural amino acids, p-aminobenzoic acid, D-biotin, calciferol, calcium D-panthotenate, cholesterol, choline chloride, folic acid, lZ069(~3 i-inositol, menadione, nicotinamide, nicotinic acid, pyridoxal, pyridoxine, riboflavin, thiamine, DL-~-tocopherol, ~een 30 (trademark of ~ao Atlas for polyoxyethylene monooleate), vitamin A, adenine, ATP, ~lP, deoxyribose, 6 ribose, glutathione, guanine, thymine, hypoxanthine, uracil, xanthine, hydrolyzate of lactalbumine, polypeptone, hydrolyzate of casein, glucose, maltose, fructose, mannitol, aextran, fumaric acid, malic acid, oxalacetic acid, citric acid, succinic acid, pyruvic acid, NaCl, KCl, l~gC12, I~gSO4,

2 4' 2 P4/ ~H2PO4, CuSO4, Fe(NO3)3, FeSO4 ~InCl (~IH4)2~1OO4, and ZnSO4. The addition of the enzymatically-decomposed animal nleat peptone in accordance with the present invention can drastically improve the yield of the plasminogen activator.
Mass culture on an industrial scale according to the present invention can be achieved by the use of roller bottle culture process, a multi-layer plate culture process, hollow fiber culture process, plastic bag culture process, and microcarrier culture process, as described in R.T. Acton and J.D. Iynn, Cell Culture and Its Application, pp. 191-216, Academic Press, `.~ew York, 1977. For mass culture on a greater scale, a microcarrier culture process is desirable.
As the peptone used in the present invention there may be employed so-called proteose peptone, protease peptone and meat peptone, which are typical bacterial culture 12069(?3 medium. The process for the preparation of the peptone is ~ell known in the art and can be performed in accord-ance with the method described in Study of Bacterial Culture ~edium, Vol. 2, by Toshikazu Sakazaki, published by Uaya Bookshop Co., Ltd., 1967. Examples of animal meats that can be used include internal organs of cattle, pig, chicken, sheep and whale, among which beef is most often used. Examples of the decomposition enz~me are trypsin, papain, pepsin and pancreatin. These animal meats are minced, mixed with water, and then adjusted with sodium carbonate or concentrated hydrochloric acid to the p~; value suitable for the enzymatic decomposition Tne pEi in the enzymatic decomposition varies depending upon tne kind of enzyme used, and it is generally 7 to 9 for trypsin, 5 to 7 papain, 2 to 4 for pepsin and 6 to for pancreatin. Then an enzyme is added to the mixture.
The mixture is then subjected to an enzymatic decomposition at a temperature of 20 to 40C for 1 to 20 days, usually at a temperature of 37C for 2 to 3 days. After being digested, the mixture is heated to a temperature of 100C
or more to inactivate the decomposition enzyme and coagulate the undigested protein. 'l'he undigested protein thus coagulated is removed by filtration, and the filtrate is concentrated, dried, and pulverized. '~he concentration, drying and pulveriæation can be achieved by boiling and ~Z06903 pulverizing or by concentrating at a low te~perature in a vacuurn device and pulverizing. ~xamples ~f commercially available peptone are Proteose Peptone, Proteose Peptone No. 2, Proteose Pep~one No. 3, and Thiopeptone from Difco, Proteose Peptone and Peptone PL 46 from Oxoid, Thiotone from BBL and Proteose Peptone from ~aigo ~iyo Kagaku Co., Lta.
The added concentration of the peptone varies depending upon the type and concentration of cells employed, 1~ and aMinO acids, vitamins, peptides, saccharides and organic acids which are allowed to coexist t?herewith, but is preferably fxom ~.1 to 4% twt/V) and more~ preferably 0.1 to 2% ~wt/v).
As described above, t~ere can be used as suitable peptone various peptones which are different in preparation method. These peptones can be used singly o~ in combination.
These peptones must be sterilized before being added to the solution. The sterilization can be achieved by directly sterilizing the peptone pow~er with ethylene oxide or y-ray, by sterilizing a solution of the peptone powder in an autoclave, or by passing a solution of the peptone powdex through a steriliza~ion filter.
The sterilization method is not specifically limited but is preferably achieved by heating at a tempe~ature of 120C in an autoclave for 10 to 6~ minutes.

-` 121~69(~3 The production of the plasminogen activator is normally carried out in 0. 2 ml or more o~ a culture liquid per 100,000 cells at a temperature of 25 to 40C, and preferably at 31 to 37C, in the pI; range o~ 6.0 to 8.0, and prefera~ly 7.0 to 7.a The maintenance of the above pH value can be achieved by tne use of a buffer system of CO2/HCO3. ~owever, if the cells produce a large amount of CO2lor organic acid such as lactic acid which obstructs the maintenance of the above p~ value, a buffer such as E'EP~S (N-2-hydroxyethylpiperazine-.~'-2-ethane sulfonic acid~ may be used. The production normally takes from 4 to 30 days, but may take more than 30 days. Since the production rate of the activator of the present invention is graaually decreased in the latter half of the production stage/ the industrial production process employs the number of days which, are the most efficient for the total production. The plasminogen activator is released from the cells into the c~lture liquid under the above.conditions.
Tne measurement of the yield of the activator was carried out as follows: .
An ayar-added fibrin plate prepared from a 95%
coagulated fibrinogen (,plasminogen content: about S0 casein unit/y coagulated protein) was used to conduct a plate methodusing urokinase as a standard r.laterial. The liquid of the plasminogen activator was diluted with 0.067 M

. lZC~69(~3 tris-~CQ buffer solution (pl~ 8.Q~ containing 1~ gelatin, O.1 M sodium chioride and 0.1~ sodium nitride. The concentration of the liquid of the activator showing the same dissolution window as 10 IU/ml of urokinase on the fibrin plate was set at 10 U/ml. When the measurement was made of the solution containing urokinase, an anti-urokinase IgG obtained from rabbits was added to the test solution so that the concentration thereof was 100 ~g/r.ll.
~hen the desired yield or time is reached, the culture liquid is collected to recover the activator.
The recovery of the Plasminogen activator can be achieved by any one of or a combination or an adsorption process, salting-out process, dialysis process, chromato-graphy process and gel filtration process, which are normally applied for recovery of protein. Examples of such purifi-cation processes are fibrin Sepharose*column chromatography using Sepharose*having fibrin bonded thereto, CM Sepharose*
column chromatography using Sepharose*having carboxymethyl group bonded thereto, lysine Sepharose*column chromato-graphy using Sepharo5e*having lysine bonded thereto, ligandexchanginy chromatography using zinc-chelated Sepharose*
lectin column chromatography using Sepharose*having Concanavalin A bonded thereto, antibody affinity chromato-graphy using antibody which specifically combines with the plasminogen activator, and gel filtration using * Trade Mark .c. ~

~Z069(D3 crosslinked dextran particles.
.~s an example of the purification process, the tissue culturP liquid is adsorbed into a CM cephalose*
column equilibrated with a 20 mM acetate buffer solution (pH 4.0~ containing 0.1% Tween*80 and 0.15 M sodium chloride. After being washed with a 20 mM acetate buffer solution (pE 4.0) containing 0.1% Tween*80 and 0.15 M
sodium chloride, the column is treated with a 20mM tris-HCQ
buffer solution (pH 8.9~ containing 0.1~ Tween*80 and 1 M
sodium chloride to effect an elution; whereby the solution of the part having the plasminogen activator activit~ is collected. The solution thus collected is dialyzed over-night at 4c a~ainst a 20 mM tris-HCQ ~uffersolution containing 0.1 M potassium rhodanide, 0.1% Tween*80, and 0.05 M
sodium chloride. The solution thus dialyzed is adsorbed into a lysine Sepharosé*COlUmn equilibrated with the same buffer solution. After being washed with an equilibrated buffer solution, the column is treated with a 20 mM tris-HCQ buffer solution containing 0.05 M sodium chloride, 1 M potassium rhodanide, 0.2 M ~-amino-n-caproic acid and 0.1% Tween*80 to effect an elution. The solution thus eluted is concentrated through hollow fibers for ultra-filtration, and then gel-filtrated through a column of Sephacry~ S-200 to obtain the desired plasminogen activator.
The physicochemical properties of the plasminogen * Trade Mark . 12~69~

activator thus obtained are illustrated below:
a) molecular weight: 63,000 + 10,000 The molecular weight measurement was effected by means of a gel filtration process using Sephadex*G-150 equilibrated with a 0.01 ~l phosphate buffer solution ~pH 7.0) containing 1.5 II sodium chloride, 0.1 M E~TA, 0.1 M arginine and 0.1% Tween*~0. Tl1e measurement of molecular weight in unreduced state by the S~S (sodi~
dodecysulphate~ electrophoresis process indicated a molecular weight of about 70,000.
b) isoelectric point: 7.0 to 8.5 . An isoelectric point electrophoresis process using ampholyte was applied to effect a fractionation at the isoelectric point, wnereby the isoelectric point was 5 measured.
c) affinity to fibrin To 950 ~1 of a 0.2~ plasminogen-free fibrinogen solution in physiological saline was added 20 ~1 of a 500 U/ml plasminogen activator solution obtained by the present invention. The admixture was allowed to stand at room temperature for 1 hour. The resulting fibrin was separated an collected from the admixture, dehydrated, and washed with physiological saline. The extraction of the activator in fibrin with 1 ml of a 2 M ammonium rhodanide solution showed ~hat about 70% of the activator had been incorporated * Trade Mark .,, ", 12069(~3 into fibrin. On the other hand, the tissue culture urokinase was not incorporated into fibrin at all.
d) affinity to Concanavalin A
2 ml of the plasminogen activator (30 U/ml) obtained by the present invention was dissolved into physiological saline. The solution thus prepared was adsorbed into a column ~0.5 x 4 cm) filled with Concanavalin A~epharose*
(manufactured by Pharmacia). The column was washed with a 1 M sodium chloride, with the result that almost 100%
of tIle plasminogen activator was adsorbed.
e) optimum pH value: 7 to 9.5 50 ~1 of the solution of the plasminogen activator in physiological saline was mixed with 50 ~1 of a 8 CU/ml -physiological saline solution of plasminogen containing 10% glycerin and 100 ~1 of various buffer solutions conta~ng a 0.10 M sodium chloride; the buffers being a 0.05 M citrate buffer solution ~pH 5.0 or 6.0), a phosphate buffer solution (pH 6.0, 7.0 or 8.0) and a glycine-sodium hydroxide buffer solution (pH 8.0, 9.0, ~0.0 or 11.0) ~i.e., seven buffers, each at a different pH of 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0).
The admixtures were preincubated at a temperature of 37C
for 30 minutes. To the admixtures thus pre-incubated were added 500 ~1 of Boc-Glu-Lys-~ys-.~CA dissolved in a 0.15 M
tris-~iCQ buffer solution (pH 8.0). The admixtures were fuxther incubated at a temperature of 37C for 15 minutes.

* Trade Mark 12~9V3 1 ml of acetic acid was added to the admixtures thus incubated to stop reaction. ~he resulting aminomethyl-cumarine was measured by fluorescence-process to determine the optimum p~ value. Tne results are shown in Fig. 1.
f) reactivity with anti-urokinase specific antibody ~abbits were immunized by tne in~ection of purifi~d urokinase (specific activity: 150,000 IU/mg protein~ with Freund's complete ad~uvant every 7 days in 35 days.
Elood was collected from the rabbits and purified to obtain 50 ~g/ml of a solution of anti-urokinase specific antiboay. The solution of the anti-urokinase specific antibody and a 20 U/ml solution of plasminogen activator obtained by the present invention were mixed with each other in a mixing ratio of 1:1. The activity of the admixture was measured according to the above mentioned method. As a result, no activity drop was recognized.
On the contrary, the urokinase activity of the mixture of the solution of the anti-urokinase antibody and a 20 IU/ml urokinase solution as a control was 100 inhibited.
As described above, the plasminogen activator obtained by the present invention does not react with anti-urokinase antibody.
The plasminogen activator thus obtained can be applied not only for medicines as thrombolytic agent but ~z069~3 also for chemicals for prevention of thrombus adapted to be bonded to artificial vein, artifioial intestines or the like and diagno~tic medicine or thrombus.
The process of the present invention is well suited for ~he stable mass production on the industrial scale of plasminogen activator which proves effective with less amount than familiar urokinase or streptokinase and has higher thrombolytic activity and less side effects than urokinase or streptokinase.

3,0 This invention will be further illustrated in the following examples:
E~PLE
500 g of well-minced beef was put into 1,000 ml of purified water. 12.5 ml of concentrated hydrochloric acid was added to the admixture. 6 g of,pepsin was then added to the ad~ixture. The admixture was allowed to effect digestion at a temperature of 37C for 2 days while being,shaken occasionally. This digested li~uid was heated to a temperature of 100C for 5 minutes. The liquid was filtered, and the filtrate was treated with sodium hydroxide to obtain pH value around neutrality (i.e., about 7.0).
While maintained at a temperature of 45C, the liquid was concentrated by means of a rotary evaporator. The liquid thus concentrated was then dried to obtain ~30 g of a light-yellowish brown pepsin-decomposed bee peptone.

~2069C?3 Ne~t, the e~fect of the pepsin-decompo~ed beef peptone over normal diploid cells derived from human fetus lung (manufactured by Flow Laboratory~ on the promotion of the production of the plasminogen activator was studied.
A plastic laboratory dish (diameter: 100 mm) was planted with the normal diploid cells in a density of 7 x 104 cells/ml. 10 ml of Medium MEM (minimum essential medium:
see Eagle H., Science, 130, 432, C1959)) containing 10%
fetal calf serum was added to the dish as a culture medium.
The dish thus prepared was then treated in air containing 5% carbon ~io~idc at a temperature o~ 37C to effect a full proliferation. The dish thus treated was washed with physiological saline. ~0 ml of ~ledium 199 containing 1%
(wt/v) of pepsin-decomposed beef peptone was added to the dish. The contents of the dish was sampled every 5 days for the measurement of the activity of the plasminogen activator.- ~he results are shown in Table 1 along with that of a control experiment free of the pepsin-decomposed beef peptone. ThR medium containing the pepsin-decomposed beef peptone showed higher yield than that free af the peptone.

lZ~69(~3 Table Yield (U~mI) 25 (day) Medium containing 8 18 32 39 47 1~ (wt/v) peptone l~edi~un free of 0,3 0,~ 1 1.2 1.5 peptone Normal diploid cells derived from human fe,us lung and human fetus foreskin tmanufactured ~y Flow Laboratory) were proliferated as in Example 1. To these proliferated diploidc,ells was added Medium MEM with different concentrations of Proteose Peptone No. 3, which is a pepsin-decomposed animal meat peptone commercially available from Difco.
The diploid cells were maintained in air containing 5%
carbon dioxide,at a temperature of 37C for 10 days.
The results of the measurement of the activity of the plasminogen activator in the culture medium are shown in Fig. 2. A rapid increase of yield appears in the peptone content range of from 1 to 4% (wt/v).
E~PLE 3 -In this exarnple, the effect of different enzymatically-decomposed animal meat peptones over normal diploid cells derived from human fetus lung or human fetus foreskin on the promotion of the production of the plasminogen activator was studied.
84 g or pepsin-decomposed whale meat peptone was prepared from 500 g of whale meat as in E.~ample 1.
110 ~ of pancreatin-decomposed beef peptone was obtained as follows:
500 g of minced beef was put into 1,000 ml of purified water. The admixture was treated with sodium carbonate to obtain a pEI value of about 8. 15 g of pancreatin was added to the admixture. The admixture was then allowed to effect digestion at a temperature of 37C
for 2 days. The liquid thus digested was treated as in Example 1 to obtain the peptone desired.
Various Medium 199 containing 1% by weight of these peptones or various commercially available enzymatically-decomposed animal meat peptones were prepared. The abovenormal diploid cells were proliferated as in Example 1.
To the diploid cells thus proliferated were added these culture mediums respectively. These culture medium were maintained in air containing 5% carbon dioxide at a temperature of 37C for 7 days to produce the plasminogen activator. The results of the measurement of the yield of the activator are shown in Table 2.
As control, the hydrolyzate of lactalbumin and polypeptone which had been not made from animal meats were used. Although these materials proved effective in the promotion of the production of the activator, the peptones made from animal meats proved far more effective.
Tabl e 2 Yield (U/ml) : Human fetus Human fetus Additive lung foreskin Nothing added 0.4 0,3 l~(wt/v) Pepsin-~ecomposed 14 3 12 6 beef peptone -1%(wt/v) Pepsin-decomposed lO 6 14 2 whale meat peptone 1%(w~/v) pancreat.in-ll 2 decomposed beef peptone 15.6 1%(wt/v) Proteose Peptone 12.4 14.4 No. 2 (Difco) 4.8 9.6 No. 3 ~Difco) 15.6 15.2 1%(w~/v) Proteose Peptone 4 9 4.6 (Daigo Elyo) 1%(wt/v) lactalbumin 2.8 2.4 hydrolyzate (Difco) 1%~wt/v) Polypeptone 4.8 5.4 (Daigo Eiyo) In this example, the effect of the pancreatin-decomposed beef peptone over various cells on the promotion of the production of the plasminoaen activator is illustrated.

~2069(~3 Specifically, ~Jarious cells were subjected to - a full proliferation in a plastic laborato~y dish (dia~.eter:100mm) according to the method of Example l. The medium of the cells thus proliferated was replaced by Medium l99 to which 1% lactalbumin hydrolyzate or 1% pancreatin-decomposed beef peptone had been added. The medium was maintained in air containing 5% carbon dioxide at a temperature of 37C for 7 days. The results of the measure-ment of the yield of the activator are shown in Table 3.
Table 3 1% pancreatin-Nothing l~ lactalbumin decomposed Cells addedhydr~lyzatebeef peptone Human fetus 1ung 0.4 14.0 25.0 human fetus 0 22.4 18.8 foreskin lS Eluman fetus 0 O.l 3.0 kidney Human fetus skin 0 0.2 3.6 Human fetus small intestines (Flow 0.2 l.6 5.4 Laboratory) A 12 liter spinner flask was planted with human fetus lung cells of a density of lO cells/ml along with Cytouex I(beads carrier for cell culture; Pharmacia's 120~g~3 registered trademark) of a concentration of 3 mg/ml.
8 liters of Medium MEM containing 10% fetal calf serum as a culture me~i~m was added to the flask. The flask thus prepared was treated in air containing 5% carbon dioxide at a temperature of 37C while being rotated at a speed of 30 rpm to effect a suspension culture. The culture was effected for 6 days so that the cells were fully proliferated. The beads carrier to which the cells had ahered was washed with physiological saline and replaced by 8 liters of Medium 1~9 containing 1% serum~free Proteose Peptone Wo. 3 (Difco). Another culture was effected with stirring at a rotation speed of 30 rpm.
The medium liquid containing the plasminogen activator of the present invention was then recovered at the time , of the replacement of the medium every 5 days.
10 liters of the,thus obtained medium liquid containing the activator at the concentration of 35 U/ml was absorbed into a CM SePharse*column (1.5~ x 10 cm) equilibrated with a 20 m~l acetate buffer solution (pH 4.0) containing 0.1% Tween*80 and 0.15 M sodium chloride.
After being washed with a 20 ~1 acetate buffer solution (pH 4.0) containing 0.1% Tween*80 and 0.15 M sodium chloride, the CM Sepharose*_olumn was treated with a 20 mM tris-~CQ
buffer solution (pH 8.9) containing 0.1% Tween*80 and 1 M
sodium chloride to effect an elution,whereby the solution - 2~ -* Trade Mark 1206~U3 of the part having the plasminogen activator activity was recovered in an amount of 103 ml. The solution thus recovered was dialyzed overnight at a temperature of 4C
against 5 liters of a 20 mM tris-HCQ buffer solution containing 0.1 M potassium rhodanide, 0.1~ Tween*80, and 0.05 ~1 sodium chloride. The solution thus dialyzed was adsorbed into a lysine Sepharose*column (2.6~ x 12 cm) equilibrated with the same buffer solution. After being washed with an equilibrated buffer solution, the lysine Sepharose*column was treated with a 20 mM tris-HC~ buffer solution containing 0.05 M sodium chloride, 1 M potassium ` rhodanide, 0.2 M ~-amino-n-caproic acid and 0.1% Tween*80 to effect an elution. 130 ml of the liguid thus eluted was concentrated to 12 ml through hollow fibers for ultra-filtration. The liquid thus concentrated was gel-filtered through a column (2.6~x 94 cm ) of Sephacryl*S-200 to recover 45 ml of the solution of the part having the plasminogen activator activity of the pr~sent invention. The plasminogen activator solution thus obtained had a concentration of

4, sao U/ml and showed a specific activity of 38,000 U/mg protein.
~Jhile the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

_ 21 _ *Trade Mark

Claims (6)

WHAT IS CLAIMED IS:

1. A process for the preparation of a plasminogen activator, which comprises bringing normal diploid cells derived from human bodies capable of producing the plasminogen activator into contact with a solution containing an enzymatically-decomposed animal meat peptone, the plasminogen activator having the following properties:
a) molecular weight: 63,000 + 10,000;
D) isoelectric point: 7,0 to 8.5;
c) affinity to fibrin: present;
d) affinity to Concanavalin A: present;
e) optimum pH: 7 to 9.5; and f) no reactivity with anti-urokinase specific antibody.

2. A process as in Claim 1, wherein the peptone concentration of said solution containing the enzymatically-decomposed animal meat peptone is from 0.1 to 4% (wt/v).

3 r A process as in Claim 1, wherein said normal diploid cells are cells derived from human lung or human foreskin.

4. A process as in Claim 1, wherein the peptone concentration of said solution containing the enzymatically-decomposed animal meat peptone is from 0.1 to 2% (wt/v).

5. A process as in Claim 2, wherein said normal diploid cells are cells derived from human lung or human foreskin.

6. A process as in Claim 4 wherein said normal diploid cells are cells derived from human lung or human foreskin.