GB2104081A - Production of plasminogen activator - Google Patents

Abstract

There is provided a process for the production of plasminogen activator (p.a.) by cultivating a diploid fibroblast strain attached to coated or uncoated surfaces, for a number of passages, in a culture medium containing sera, and cultivating the conditioned cells in a culture medium to produce p.a., which is recovered from the media.

Description

SPECIFICATION Production of plasminogen activator The invention relates to the production of plasminogen activator, (p.a.), by a special process of cultivating certain cells. According to the invention a novel process of cultivation is provided, as well as certain cell lines which are characterized by a high production of p.a., without transformation with virus or treatment with chemicals (inducers) which are carcinogenic or cocarcinogens. At the end of the production cycle the cells are unchanged as to their number of chromosomes and growth characteristics. They proliferate under the standard conditions used for each cell line and thus can be recycled.

BACKGROUND OF THE INVENTION Urokinase is a proteolytic enzyme which is used in medicine for the treatment of patients suffering from blood clots. Its efficacy has been proved, and its use is permitted according to the regulations of the F.D.A. (USA), as well as in Europe and Japan. It seems that the enzymes designated as urokinase and as plasminogen activator are essentially identical. Urokinase is produced nowadays mainly from urine wherein it is present in very small quantities (of the order of 8 units/ml). The cost of the final preparation is very high, and the available quantities are small. As a consequence, efforts are going on to produce p.a. in commercial quantities by alternative methods.

SUMMARY OF THE INVENTION The invention relates to a process for the production of plasminogen activator, hereinafter p.a., and to the thus produced enzyme. The invention is based on the unexpected discovery that the said enzyme p.a. can be produced in commercially attractive quantities from normal diploid cells.Cells were isolated which produce p.a., and such cell lines were conditioned to produce increased quantities of p.a., as set out in detail hereinafter It was found that certain cell lines of established human diploid cells, such as IMR-90, WI-38 or HEL-299 acquire the ability to produce p.a. when same are grown initially for a certain period of about 5-15 days, on a medium containing a serum other than fetal calf serum, preferably horse serum, or by growing them on dishes precoated with a matrix, such as poly-D-lysine. Cells thus conditioned, produce afterwards and excrete p.a. continuously for a period of at least 1 5 days in a medium devoid of serum, containing lactalbumine hydrolysate or another suitable nutrient medium.After accumulation of p.a. in the medium, and when a certain concentration is reached, p.a. production decreases, and eventually even stops. The value of the maximum concentration was about 1 U/ml for many normal cell lines; 3-5 U/ml for transformed cells, 30-50 U/ml for porcine bladder cells and 100--300 U/ml for IMR-90. Continuous removal of p.a. from the nutrient medium prevents the attainment of the maximum concentration, and thus the cells produce p.a. for a considerably prolonged period of time. With IMR-90 this rate is about 50 U/day/l 05 cells. The p.a. can also be absorbed from the medium and same can be recycled. The p.a. produced was analyzed and found to have a M.W. of about 55 KD, it cross-reacts immunologically with urinary UK.It thus seems that the p.a. produced is homologous to the 54 KD species of urinary urokinase (H-UK) which seems to be the species having the highest therapeutic efficacy.

IMR-90 is a cell line similar to Wl-38 which latter is an approved source for the production of live polio and rubelia vaccines. The cultivation of these cell lines is described in detail in the literature, and the optimum conditions are well known. According to the present invention, the process is described with reference to the IMR-90 cell line. It ought to be understood that this is by way of example only and that similar established, or novel cell lines having similar characteristics can be used for the same purpose. It is to be understood that the following description is by way of illustration only.

DESCRIPTION OF THE PREFERRED EMBODIMENT The invention relates to the production of plasminogen activator (p.a.) by cultivating certain diploid cells under defined conditions, and recovering the desired product from the cultivation medium.

A preferred cell line used is IMR-90, a human diploid fibroplast strain derived by W. W. Nichols (1977) from the lung of a 16 week old female fetus. The cells were obtained from ATCC (CCL 186, passage 7). Neither IMR-90 nor WI-38 nor HEL-299 have been reported to produce p.a. in an appreciable quantity. In the present research they were initially considered as non-producers. It has been discovered that it is possible to convert such cells to producers of appreciable quantities of p.a. by cultivating same on a medium containing horse serum, and by the subsequent cultivation on other nutrient media. The IMR-90 cells received from ATCC were thawed and plated in a medium containing 10% FCS as recommended by ATCC. After one passage the cells were transferred to the LH medium and enzyme content was assayed.A slow formation of p.a. was observed (less than 5 U/ml after 4 days).

After two passages in FCS the IMR-90 cells were subcultured in a medium containing 10% HS.

After 4 passages (estimated 1 2 doublings) in this medium, the cells were transferred to LH. The results (Fig. 1) showed that the cells became very high producers of p.a. The enzyme was produced rapidly and its concentration in solution reached about 100 U/ml.

The same cells were then continuously subcultured in HS and p.a. production was retested from time to time. The results (Table I) indicated that the capacity to produce p.a. was retained until the cells started to show the signs of old age, i.e. slow division and "flattening".

This was observed after passage 22-24, which is estimated to correspond to 65-70 doublings.

To eliminate the possibility that p.a. production resulted from a singular and irreproducible mutational event, the experiment was repeated starting from cells frozen after passage 9. The results showed that the capacity to produce p.a. is reproducibly expressed.

Under certain conditions, the above fibroblast cell lines can be conditioned to produce p.a. by cultivation on certain substrates in the presence of sera. Cells were grown on a variety of sera, using culture vessels which were precoated with various compounds (see below) and the appearance of the capacity to produce p.a. was followed. The amount of p.a. in the medium, was found to vary as function both of the serum, and of the matrix. The results obtained with vessels precoated with poly-D-lysine show (Table IV) that on this matrix cells can be adapted to produce p.a. in media (such as FCS) which otherwise are not conducive to enzyme production. Once adapted these cells continued to produce the enzyme in a medium devoid of serum as described above. The conditioned cells are used in the production process.

Following the observations described above, the production was characterized in some detail. In these experiments the cells were grown in a medium containing HS, then transferred to a medium containing LH, and the level of p.a. in the medium was monitored.

The results summarized in Fig. 2 show that with cells from early passages the enzyme concentration in the medium reaches an upper limit of about 100 U/ml. This upper limit increases when cells from later passages are used. With very old cells enzyme production did not cease even when its concentration in the medium reached 300 U/ml.

The results strongly suggest that the p.a. level observed in the medium is determined by its formation on one hand and some "negative" process on the other. The latter could be a repressive feedback on the synthesis (or excretion) of the enzyme or the formation of another cell product which either inhibits the enzyme or catalyzes its decay. Repeated passaging of the cells in a medium containing HS seems to decrease this negative control, thereby allowing the buildup of higher detactable enzymic activity in the medium.

It should be pointed out that an activity of 100 U/mi corresponds to an enzyme concentration of about 1 mg/lit, so that the product levels in the above experiments are in the range of 1-3 mg/lit.

HARVESTING AND PURIFICATION The p.a. produced by the cells can be harvested by adsorption from the culture medium using adsorbents known to adsorb urokinase, by concentration using membranes, or by combinations of the above. Two examples are given by way of illustration only.

1. Adsorption The LH medium containing p.a. was treated with finely divided silica, by adding 1-2 mg of solids to each ml of solution, and mixed for 1 hour. The solids were then recovered by centrifugation. The supernatant was found to contain less than 5% of the p.a. present initially and was reused as described below.

Analysis of the solid revealed that p.a. could be quantitatively released from the silica by treatment with a base, particularly ammonia, organic amine and polymeric amines to yield a solution of p.a. which could then be further purified by a variety of methods employed in this field.

2. Dialysis-concentration The LH medium containing p.a. was transferred to a dialysis bag and a water absorbing material put on the outside. The enzyme was thereby concentrated 1 0-20 fold with a total recovery better than 75%.

Effect of medium Changes on p.a. Production The similarity between the time course of p.a. production by IMR-90 and those observed previously with other cells, led to tests of the effect of repeated medium changes. Like the previous cases, it was found that (Fig. 2) that the slow-down in enzyme appearance in cultures of IMR-90 at high enzyme concentration is abolished to a large extent by perfusion. Apparently, perfusion somehow interferes with the negative "feedback" control mechanism discussed above.

The results in Fig. 3 seem to indicate that changing the medium affects the rate of appearance of p.a. when an appreciable amount of enzyme accumulates in the medium but does not affect the initial rate of p.a. production. Supporting evidence was obtained from an experiment in which cells grown in FCS were transferred to LH (Fig. 4). These cells produced p.a. very slowly, so that the concentration in the medium was low, and the rate of p.a. formation was not effected by medium changes.

Since the cost of medium is expected to be an important economic factor an experiment designed to test the possibility of medium recycling was carried out. The spent medium was treated with silica to remove p.a., and returned after 1:1 dilution with fresh medium. The treatment with silica was found to remove 90% or more of the p.a. from the medium. The results (Fig. 5) indicate that medium recycling is possible. The observed production of p.a. was actually higher with recycled medium than on perfusion with fresh medium.

The medium used for the production of p.a. in the above experiments contained lactalbumin hydrolysate, following the general procedures used in investigations of p.a. It was found however that p.a. production can be obtained with a variety of peptones used as substrates for the growth of microorganisms and which are the hydrolysis products of proteins of animal or plant origin. A sample of the results is shown in Table V.

THE CULTURE OF IMR-90 CELLS 1. Standard Procedure Cells were grown routinely in Falcon 90 mm plastic plates, in a medium composed of a mixture (1/1 v/v) of DMEM and F-i 2 and either fetal calf or horse serum (purchased from Gibco USA) were added to an actual concentration of 10%. Each plate was seeded with 5 x 105 cells and 7 ml of medium.

Confluence was reached in 3-5 days at about 8 x 106 cells per plate. For passaging the cells were trypsinized with 0.25% trypsin solution (Biolab, Jerusalem). The trypsin solution (2-3 ml) was added to the plate, and after 1 minute at room temperature the bulk of the trypsin solution was aspirated. The cells were left covered with a thin film of liquid. After 1 5-20 minutes a small amount of fresh medium was added, mixed with the cells and drawn into a pipette several times to obtain a suspension of dispersed cells. Fresh medium (5 ml) was then added to each plate and aliquots were taken for counting cells and for replating.

Cells were also grown on plates of other sizes, or in other vessels used for cell culture, using the ratio of cells to surface area as above and following the same general procedure.

2. Effects of Substrate on Attachment and Proliferation of IMR-90 Studies on cultivation of IMR-90 on various substrates and coatings were carried out. The cells were plated and allowed to grow on various substrates for 4 days in order to obtain a combined effect on plating efficiency and proliferation.

The results (Tables II and Ill) show that cultivation can be improved by precoating the plates. The simplest effective precoat is gelatine. Furthermore, a gelatine coat also enables growth on other surfaces such as Gelbond.

Units Enzyme activity is expressed in CTA units.

Enzyme assays were performed by the fibrin plate method calibrated with standard urokinase obtained from the NIH (USA).

CHARACTERIZATION OF CELLS AFTER USE FOR p.a. PRODUCTION IMR-90 cells adapted for p.a. production as described above were karotyped. A normal complement of chromosomes (2N = 46) was found with a distribution of experimental results similar to that reported from the original cell line.

Cells for the production of p.a. in LH medium for 12 days, were transferred into a growth medium containing 10% FCS, and propagated under the standard conditions described above. The cells were followed for 1 month, and it was observed that the growth rate, density of cells at confluence and morphological appearance of the cells were identical to the original IMR-9O. The p.a. production also dropped to its level of normal cells.

Similar results can be obtained with cells of cells lines such as WI-38, HEL-299 or the like and the invention is intended to extend also to the use of such cell lines to the the production of p.a.

TABLE I PRODUCTION OF p.a. BY IMR-90 CELLS PROPAGATED IN A MEDIUM CONTAINING HORSE SERUM Exp. Passage Number* p.a. level U/ml 1 12 98 15 110 20 208 22 45** 24 15.6 2 14 160 1 7 43** 24 272 * Passages numbered from 8 on.

** Only 0.5 x 105 cells.

The cells were continuously subcultured in HS medium, by the standard procedure. To determine p.a. production the cells were plated, after trypsinization, in HS medium, (2 x 105 cells, 30 mm plate, 1.5 ml medium) and 24 hours later the medium was changed to LH. Activity was determined on an aliquot of the medium 96 hours after transfer to LH. Blanks, runs with plasminogen-free plates were negative.

IMR-90, received frozen after passage 7, were thawed and passaged twice (passage 8 and 9) in DMEM/FCS. The first experiment was carried out with cells frozen after passage 9 and stored in liquid N2. The cells were thawed, passaged twice in DMEM/FCS, then passaged in DMEM/HS.

TABLE II THE USE OF GELBOND* FILMS AND GELATINE COATING FOR CULTIVATION OF IMR-90 Substrate Cell count ( x 10-5) Untreated plate 1.3 Gelatine treated plate 3.2 Gelbond 1.0 Gelatine treated Gelbond 1.7 * Gelbond is a trade name (Marine Biocaloids) for thin sheets of polyester. One side of the sheet has been made hydrophilic by an undisclosed process, presumably with some alkali. The hydrophilic side was used.

The experiments were carried out in 30 mm plates, starting with 1 x 105 cells, and 1.5 ml HS-medium. For experiments with Gelbond a circle of the film was prepared and covered the bottom of the plate. Gelatine was applied as an 0.03% aqueous solution, left 20 min. in the plate and the solution was removed by aspiration.

After 96 hours of growth the cells were detached by trypsinization and counted.

TABLE ill THE USE OF VARIOUS COATING FOR PLATES Substrate Cell count (x 10-4) N/No.

Untreated plate 4.5 1.3 Treated protamine sulfate 3.0 .8 Treated poly-D-lysine 6.5 1.8 Cell matrix 7.5 2.1 The experimental details are as in Table li, except that a different batch of cells was used and 2.6 x 104 cells were plated in 30 mm plates. The plates were treated with protamine sulfate or poly-Dlysine (0.1 mg/ml).

After 5 minutes incubation, the plates were washed twice with sterile H20 and once with medium, and the cells were plated.

The cell matrix was prepared by growing monolayers of CCL6 cells (human intestine, epithelial) and treatment with 0.5% triton x-1 00 in H2O. After 10 minutes incubation the plates were washed twice with PBS and once with medium. At this stage the plates were ready for plating cells.

TABLE IV PRODUCTION OF p.a. BY IMR-90 CELLS PROPAGATED IN VARIOUS SERA Poly D-lysine p.a. in the medium Serum coating U/ml Fetal calf serum + 23 Fetal calf serum 5 Horse serum + 57 Horse serum 28 Porcine serum + 3 Procine serum 1 Human cord serum + 6 Human cord serum - 11 New born calf serum + 40 New born calf serum - 10 Dog serum + 12 Dog serum 8 Goat serum + 8 Goat serum 3 Chicken serum + 3 Chicken serum 2 IMR-90 cells were propagated in a medium containing 10% of serum, on plastic petri dishes precoated with 0.1% poly-D-lysine. The p.a. activity was assayed in the medium.

TABLE V EFFECT OF VARIOUS PEPTONES ON p.a. PRODUCTION p.a. in the medium Peptone U/ml Lactalbumin hydrolysate 36 Casein hydrolyzate 72 D.M. peptone 27 Tryptose phosphate broth 10 Prematone-K 11 Cells were plated (1 or cells/30 mm dish) in a medium plus 10% HS, and 48 hrs later the medium was changed to fresh medium with the indicated peptone. The p.a. in the medium was assayed after 24 hrs.

Lactalbumin hydrolysate 0.5% from Bio-lab ISRAEL Casein hydrolysate 2% from Gibco U.S.A.

D.M. Peptone 0.5% from Scientific Protein Labor. Inc.

Tryptose phosphate broth 0.5% from Difco Prematone-K 0.5% from Humko Sheffield Chemicals NOTES ON THE ACCOMPANYING DRAWINGS Fig. 1 shows the production of p.a. by IMR-90 cells propagated in HS medium. The cells were propagated four passages (after passage 8) in a medium containing horse serus (HS).

Fig. 2 shows the time course of p.a. production by IMR-90 cultivated for various times in DMEM/HS.

Fig. 3 shows the effect of medium changes on p.a. production by IMR-90 cells, propagated in HS medium. IMR-90 cells after passage 15 (6 passages in HS) were plated (1 x 105) in 60 mm petridishes with 2.5 mls of HS medium. 24 hrs. later the medium was changed to LH. At the times indicated by arrows, half or total medium in each plate was harvested and replaced with fresh LH medium. In the control plates 100 it of medium were harvested for p.a. assay and replaced with fresh LH medium.

Fig. 4 shows the effect of medium changes on p.a. production by IMR-90 cells, propagated in FCS medium. IMR-90 cells after passage 9 in FCS, were plated (5 x 104) in a well (16.4 mm) with 2 mls of a medium containing FCS. 24 hrs. later the medium was changed to one containing LH, and the experiment continued.

Fig. 5 shows the continuous production of p.a. by IMR-90 cell IMR-90 cells after passage 23 (13 passages in HS medium) 2 x 105 cells were plated in 60 mm Petri dishes with 2.5 ml of medium containing HS. 24 hrs. later the medium was changed to LH. Media were changed at the times indicated by arrows. In group 1 half of the medium in each plate was harvested and replaced with fresh LH medium. In group 2 the total medium was harvested. 1.25 mls of fresh LH medium were added to the cells. The harvested medium was treated with silica (to adsorb p.a.) and 1.25 ml of this medium was added back to each plate of group 2. P.a. activity was determined on aliquots of medium at each point.

Claims (11)

1. A process for the production of plasminogen activator (p.a.) which comprises cultivating a diploid fibroblast strain attached to coated or uncoated surfaces for a number of passages in a culture medium containg sera, and cultivating the conditioned cells in a culture medium to produce p.a., which is recovered from the media.

2. A process according to claim 1 wherein the diploid fibroblast cells are of the IMR-90, Wl-38 or HEL-299 cell lines.

3. A process according to claim 1 or 2 wherein the cells are cultivated on the uncoated surface of plates, sheets, beads or other surface configurations.

4. A process according to any of claims 1 to 3 wherein the cells are conditioned by cultivation on surfaces coated with poly-D-lysine, protamine sulfate, gelatine or a cellular matrix and subsequently cultured on an uncoated or coated surface.

5. A process according to any of claims 1 to 4 wherein the cells are conditioned by cultivation in a medium containing horse serum (HS) for 3 to 12 days, and subsequently cultivated in a medium containing lactalbumine hydrolysate (LH) to produce the desired p.a.

6. A process according to any of claims 1 to 5 wherein the cells are conditioned to produce p.a. by cultivation in a medium containing fetal calf serum, porcine serum, human cord serum, newborn calf serum, dog serum, goat serum or chicken serum, and subsequently cultivated in a medium containing a suitable hydrolysate to produce the desired p.a.

7. A process according to claim 6 wherein p.a. production is carried out by cultivation in a medium containing a peptone selected from caseine hydrolysate, D.M. peptone tryptose phosphate broth, Prematone-K or other commercially available equivalents.

8. A process according to any of claims 1 to 6 wherein the produced p.a. is continuously removed from the culture by perfusion.

9. A process according to any of claims 1 to 8 wherein the p.a. is adsorbed on silica, released by contact with a base and purified by conventional means.

1 0. A process according to claim 1 wherein the medium is recycled after removal of p.a.

11. Process for the production of plasminogen activator, substantially as hereinbefore described.

1 2. Plasminogen activator whenever obtained by a process according to any of claims 1 to 11.