FR2510604A1 - PRODUCTION OF PLASMINOGEN ACTIVATOR - Google Patents

Abstract

PLASMINOGENE ACTIVATOR PRODUCTION PROCESS (A.P.). IT CONSISTS OF CONDITIONING DIPLOID FIBROBLAST CELLS, FIXED ON COATED OR UNCOATED SURFACES, BY A CERTAIN NUMBER OF PASSAGES IN A CULTURE ENVIRONMENT CONTAINING SERUM, THEN IN CULTIVATING THE CELLS THEREFORE CONDITIONED TO CULTIVATE IN AN APURE. THUS OBTAINS, FROM NORMAL CELLS, GREATER QUANTITIES OF PA

Description

The present invention relates to a method of

  plasminogen activator, hereinafter referred to as

  a.p, as well as the enzyme thus obtained.

  Urokinase is a proteolytic enzyme, used in medicine to treat patients with blood clots Its effectiveness has been proven, and its use

  is allowed to both U S A, Europe and Japan.

  pon It seems that the enzymes, designated under the names

  urokinase, on the one hand, and plasminogen activator from

  on the other hand, are virtually identical at present

  the, urokinase is produced mainly from 1 '

  urine, in which it is present in very small quantities

  The cost of the final preparation is very high, and the quantities available are very low. Consequently, we have tried to produce p. by other processes, in quantities that can be

trade.

  * The present invention is based on the observation

  unexpectedly that the enzyme p could be obtained in

  industrially interesting quantities, from

  normal diploid cells Cells are isolated, which produce p, and their lines are conditioned to produce increased amounts of p, without resorting to

  transformations by means of viruses or chemical treatments

  (inducing agents) which are carcinogenic or co-carcinogenic

  At the end of the production cycle, the cells are

  changes in the number of their chromosomes and in their growth characteristics They proliferate in

  normal conditions, used for each line of

  lules and can be recycled.

  The new process according to the invention,

  a p, consists in cultivating a diploid fibroblast strain, fixed on coated or uncoated surfaces, for a number of passages in a culture medium.

  containing serum, then to grow the cells thus con-

  in the culture medium where they produce

  from the p, which is recovered from this medium.

  It has been found that certain established human diploid cell lines, such as IMR-90, WI-38 or HEL-299, acquire the ability to produce α p when initially cultured for a certain period of time. order of 5 to 15 days, on a medium containing a

  serum other than fetal calf serum (FCS), preferably

  horse serum (HS), or when cultured on cups previously coated with a matrix, as

  poly-D-lysine The cells, thus conditioned, produce

  subsequently, and excrete the p continuously for a period of at least 15 days, in a serum-free medium containing lactalbumin hydrolyzate (LH) or other suitable nutrient medium. p in the middle, and when a certain concentration

  is reached, the production of a p decreases, and even

  the value of this maximum concentration

  male is of the order of 1 U / ml for many

  normal cells; from 3 to 5 U / ml for trans-cells

  formed from 30 to 50 U / ml for the bladder cells of

  pork and from 100 to 300 U / ml for IMR-90

  of the nutrient medium, prevents this from occurring

  the maximum concentration; thus, cells produce a p for a very long time with IMR-90

  the production rate is about 50 U / day / 105 cells.

  The p can also be absorbed from the medium and the medium can be recycled. The product is analyzed and found to have a molecular weight of about 55 KD, and to have immunologically cross-reactive reactions. with urinary urokinase (UK) So it seems that the p product

  and the 54 KD urinary urokinase (H-UK) species are homologous

  the latter being considered as the species

  with the strongest therapeutic activity.

  IMR-90 is a cell line similar to WI-38, the latter being a classic source for

  production of live vaccines against polio and rubella

  The culture of these cell lines is described in detail in the literature, and the optimum conditions are well known in accordance with the invention.

  is described with reference to the IMR-90 cell line.

  It is understood that this is only done as an example

  non-limiting, and that one can use, in the same

  purpose, new or established cell lines, similar to

  with similar characteristics.

  Among the various sera that can be used according to the invention, mention may be made of those of the pig, the human cord, the newborn calf, the dog, the goat, the chicken, etc. A preferred cell line used is

  IMR-90, a strain of human diploid fibroblasts, ob-

  held by WW Nichols (1977) from 16 week old female fetus lung Cells from ATCC (CCL 186, passage 7) No more IMR-90, than WI-38 or HEL-299 had the reputation of producing ap in appreciable quantity Originally, during the present research, these strains were considered as nonproductive It was discovered that it was possible to convert these cells

  producers of appreciable quantities of a p, by

  on a medium containing horse serum, followed by

  subsequent culture on other nutrient media.

  IMR-90 cells from ATCC are thawed and plated in medium containing 10% FCS, as recommended by ATCC. After one pass, the cells are transferred to LH medium, and the enzyme content is assayed.

  slow formation of p (less than 5 U / ml after 4 days).

  After two passages in FCS, the IMR-90 cells are subcultured in medium containing 10% HS (horse serum). After four passages (12 duplications estimated) in this medium, the cells are transferred to LH The results (FIG. 1) show that the cells become

  very strongly producing the p The enzyme is

  quickly and its concentration in the solution-at-

dyes about 100 U / ml.

  In FIG. 1, on the ordinate, is carried the activity of the a.p in U / ml and, in abscissa, the duration in days of the stay.

in the middle LH.

  The same cells are subcultured continuously in HS and the production of a p is controlled from time to time.

  The results (Table 1) indicate that the

  the city of production of the p remains, until the cells begin to show signs of aging,

  that is to say, a slow division and flattening ".

  This phenomenon is observed after the passage 22 to 24, which

  is estimated to correspond to 65 to 70 duplications.

  In order to eliminate the possibility that the production of a p is the result of a case of singular mutation

  and not reproducible, we repeat the experiment in par-

  so many cells frozen after the ninth pass The results

  States indicate that the production capacity of the a p is

premium in a reproducible manner.

  Under certain conditions, the above fibroblast cell lines can be conditioned to produce p, by culturing on certain substrates.

  presence of sera The cells are grown on different

  sera, in culture vessels which have been previously coated with various compounds (see below), and the appearance of the production capacity of the ap is observed. It is found that the quantity of a p in the middle

  varies both serum and matrix.

  results obtained with containers previously coated with poly-Dlysine (Table IV) show that, on these matrices, cells can adapt to produce a p in

  media such as FCS (fetal calf serum) which other-

  do not lead to enzyme production.

  These cells continue to produce the enzyme in a serum-free medium as described above.

  packaged are used in the production process.

  tion. According to the observations described above, the production is characterized by a few details During these experiments, the cells grow in a medium containing HS, then are transferred to a medium containing LH, and the level of α p in the medium.

is controlled.

  The results, shown in Figure 2, show

  trent with cells from early passages,

  the concentration of enzymes in the medium reaches a level

  mite of the order of 100 U / ml This upper limit

  increases when using cells from later passages With very old cells

  enzyme production does not stop, even when its concentration

  in the medium reaches 300 U / ml.

  In FIGS. 3 to 5, the following signs are used: O for the whole of the replaced medium; A replacement of half of the medium;

* no replacement at all.

  Figure 3: The cells IMR-90, after passage 15 (6 passages in HS) are deposited (1x O) in 60 mm Petri dishes with 2.5 ml of HS medium 24 hours later this medium is replaced by LH At the times indicated by the arrows, half or all of the medium in

  each box, is collected and replaced with fresh LH.

  In the control boxes, 100 μl of medium are collected

  to try a p and are replaced by fresh LH.

  Figure 4: The IMR-90 cells, after passage 9 in FCS, are plated (5 x 10) in a housing (16.4 mm) with 2 ml of medium containing FCS 24 hours later.

  medium is replaced by a medium containing LH, and it is

follow the experience.

  FIG. 5: The IMR-90 cells, after the passage 23 (13 passages in ES medium), are deposited (2 × 10) in the Petri dishes of 60 mm with 2.5 ml of medium containing HS 24 hours later, this medium is replaced by LH The media are changed at the times indicated by the arrows. In group 1, half of the medium in each dish is collected and replaced with fresh LH medium. In group 2, the entire medium is collected. 1.25 ml of fresh LH is added to the cells. The harvested medium is treated with silica (to absorb ap) and 1.5 ml of

  this environment is recycled to each group 2 box.

  tivity of a p is determined on half-life samples.

place in each point.

  These results strongly suggest that the rate

  of a p, observed in the medium, is determined by its forma-

  This may be an inhibitory feedback on the synthesis (or excretion) of the enzyme, or the formation of

  another cellular product that inhibits the enzyme or cata-

  lysis degeneration The repeated passage of these cells, in a medium containing ES, seems to lessen this setting

  negative, thus allowing the accumulation of activity

  zymatic detectable, superior, in the middle.

  It should be noted that an activity of 100 U / ml corresponds to an enzyme concentration of the order of 1 mg / liter,

  so that the product rates in the experiments

  above are in the range of 1 to 3 mg / liter.

  Harvesting and purification of the product is

  proceed as follows: the p produced by the cells can be harvested by adsorption from the culture medium,

  using known agents to adsorb urokinase, by means of

  centration by means of membranes, or by combination of these

  Two examples are given below as examples of

non-limiting lustration.

1 Adsorption.

  The LH medium containing the p is treated with finely divided silica, by adding 1 to 2 mg of -olides per ml of solution, and mixing for 1 hour the solids are then recovered by centrifugation.

  the supernatant contains less than 5% of the initial

  tially present, and reused as described more

far -

  The analysis of the solid reveals that a p can be quantitative-

  silica, organic amine and polymeric amines, which gives a solution of the

  purified by various methods used in this field.

  2 Dialysis concentration The p-containing LH medium is transferred to a dialysis bag and a substance is placed on the outside.

  absorbing water The enzyme is thus concentrated from 10 to 20-

  times, with total recovery greater than 75%.

  We study the action of environmental modifications

  on the production of a p in the following way.

  The similarity between the production times of the a p by

  IMR-90 and those that have been observed in the past with

  very large cells, leads to tests on the exchange rate-

  There is evidence (Figure 2) that, as

  in previous cases, the delay in the appearance of

  zyme in cultures of IMR-90, with a high concentration of

  enzyme, is abolished to a great extent by an infusion.

  Apparently, the infusion somewhat interferes with the negative feedback control mechanism that it has

been discussed above.

  The results of Figure 3 (ordinate the pro-

  total enzyme production expressed in units and abscissae in the number of days in LH medium) suggest that the change in the medium affects the rate of onset of AP when an appreciable amount of enzyme accumulates in the middle but does not act on the initial velocity

  A further proof is provided by the

  experiment, in the course of which

  lules, having grown in FCS, are transferred to LH (

  Figure 4: on the ordinate the total production of enzyme expressed in Unity, and in abscissa the number of days in the

  medium LH) These cells produce a very slow p

  ment, so that the concentration in the medium is

  weak, and the speed of formation of a p is not affected

by changes in the environment.

  Since the cost of the environment is considered an important economic factor, an experiment is carried out

  designed to try the possibility of recycling this medium.

  The spent medium is treated with silica to remove p, and it is recycled after dilution in the proportion of 1/1 with fresh medium. It is found that the treatment with silica eliminates 90% or more of the p content in

  the middle The results (Figure 5: on the y-axis the produc-

  total enzyme and in abscissa the number of days in

  medium LH) indicate that the recycling of the medium is pos-

  The observed production of a p is actually higher with the recycled medium than during the infusion of

fresh medium.

  The medium used for the production of the p,

  during the above experiments, contains hydro-

  lactalbumin (LH) lysate, following the general procedure

  used in research on p We observe, however,

  that the production of a p can be obtained with different peptones, as a substrate for the growth of

  microorganisms which are products of hydrolysis of

  The results are given in Table V. Culture of IMR-90 Cells 1 Normal Procedure The cells are cultured in the usual manner on Falcon plates of plastic material, of 90 mm, in a medium. composed of a mixture (1/1 in volume / volume) of DMEM and F-12, and to a real concentration of%, fetal calf serum (FCS) or horse serum (HS), provided by Gibco USA Each plate is seeded with 5 x 105 cells and 7 ml of medium. The confluence is

  3 to 5 days, at approximately 8 x 106 cells per

  that for their passage, the cells are trypsinized

  with 0.25% trypsin solution (Biolab, Jerusalem).

  2 to 3 ml of trypsin solution are added to the plate, and

  after 1 minute at room temperature, we aspire to this

  The cells are left covered with a thin layer of liquid After 15 to 20 minutes, a small amount of fresh medium is added, mixed with the cells and aspirated into a pipette with several layers of liquid.

  times, to obtain a suspension of disper-

  5 ml of fresh medium are then added to each plate, and portions are taken to count the cells and

to redeposit them.

  The cells were also cultured on plates of

  different cells, or in other containers used for cell cultures, using the same cell / surface area ratio as above, and following the

same general procedure.

  2 Substrate effects on the binding and proliferation of IMR-90 Studies on the culture of IMR-90 have been conducted

  on different substrates and coatings The cells are de-

  placed on different substrates and allowed to proliferate for 4 days so as to obtain a combined effect on 1 '

  deposit efficiency and proliferation.

  The results (Tables II and III) show that culture

  can be improved by pre-coating the plates.

  The simplest effective precoating consists of gelatin In addition, a layer of gelatin allows

  also growth on other surfaces like "Gelbond".

  Units The activity of the enzyme is expressed in units CTA The enzyme tests are carried out by the process of the fibrin plate, graduated with normal urokinase

from NIH (USA).

  Characterization of cells after use for the production of a p.

  IMR-90 cells, adapted for the production of ap.

  as described above, are karyotyped A complement

  chromosome normalization (2 N = 46) is observed, with

  Experimental results, similar to that of the original cell line Cells, used for the production of p in LH medium for 12 days, are transferred into growth medium containing% FCS, and propagated in the normal conditions, described above These cells are tracked during 1

  months, and we observe that, the speed of growth, the den-

  Cellularity at the confluence and morphological aspect

  cells, are identical to those of the original IMR-90.

  The production of a p also falls to the level of that

normal cells.

  Similar results can be obtained with

  cells of lines such as WI-38, HEL-299, or

  and the invention also relates to the use of

  of these cell lines for the production of a p.

TABLE I

  Production of a p by IMR-90 cells, which propagate in a medium containing horse serum (HS) Exp Number of Rate of a p U / ml passages *

1 12 98

110

208

22 45 **

24 15.6

  Exp Number of Rates of a p U / ml passages *

2 14 160

17 43 **

24,272

  Passages numbered from 8 * 0.5 x 10 5 cells, only The cells are subcultured continuously in HS medium (horse serum), by the normal procedure.

  determine the production of a p, they are deposited,

  near trypsinization, in HS medium, (2 x 10 5 cells, 30 mm plate, 1.5 ml of medium) and 24 hours later

  this medium is replaced by LH (hydrolyzate of lactalu-

  mine) Activity is determined on a portion of this half

  96 hours after transfer to LH.

  with plasminogen free plates are nega-

  tive. IMR-90, received frozen after passage 7, snt thawed

  and passed twice (passages 8 and 9) in DMEM / FCS.

  The first experiment is conducted with

  frozen after passage 9, and stored in free nitrogen

  These cells are thawed, passed twice

  in DMEM / FCS, then switched to DMEM / HS.

TABLE II

  Using GELBOND * Films and GELATINE Coating to Grow IMR-90 Substrate Cell Count (x 10-5 Untreated Plate 1, 3

Plate treated with

  Latin 3,2 Gelbond 1,0 gelbond gelatin treated 1,7 * Gelbond is the brand name (Marine Biocaloids) thin polyester sheets One side of the sheet is made hydrophilic by a method not described, probably to the help

  This is the face we use.

  The experiments are carried out on plates of

  mm, starting with 1 x 105 cells and 1.5 ml of medium

  HS For experiments with "Gelbond", we prepare

  a round of film and cover the bottom of the plate.

  Gelatin is applied as an aqueous solution

  0.03%, left on the plate for 20 minutes, and then

The solution is removed by aspiration.

  At 4 out of 96 hours of growth, the cells are removed.

  trypsinized, and counted.

* TABLE III

  Use of various plate coatings Substrate 4 N / cell counts (x 10) Untreated plate 4.5 1,3 Protamine sulfate treated 3.0 0.8

Treated with poly-D-

  lysine 6.5 1.8 cell matrix 7.5 2-, 1

  The experimental details are the same as for the

  except that a different series of cells are used and 2.6 x 104 cells are plated on 30 mm plates. These plates are treated with sulphate.

  protamine or poly-D-lysine (0.1 mg / ml).

  After 5 minutes of incubation, the plates are rinsed twice with sterile H 2 and once with medium.

and the cells are deposited therein.

  The cell matrix is prepared by the growth of

  monolayers of CCL 6 cells (epithelial, human intestine

  hand) and treatment with "Triton X-100" at 0.5% in water.

  After 10 minutes of incubation, the plates are rinsed

  twice with PBS and once with medium.

  At this point, the plates are ready to receive cells.

TABLE IV

  Production of a p by IMR-90 cells

  gent in various serums Serum Coating of AP in Fetal Calf Serum (FCS) Horse Serum (HS) i Pig Serum II Serum of Human Cord II Serum of Newborn Calf II Serum of Dog It Serum of Goat Serum of chicken he Poly D-lysine + + + + + + + + medium in U / ml i IMR-90 cells spread in a medium containing

  % of serum, in plastic Petri dishes

  than previously coated with 0.1% poly-D-lysine.

  The activity of a p is tried in the medium.

TABLE V

  Effect of different peptones on the production of a p. Peptone ap in the medium U / ml Lactalbumin hydrolyzate 36 Casein hydrolyzate 72 D. M peptone Tryptose phosphate broth Prematone K Lactin albumin hydrolyzate DM peptone 1 1 0.5% Bio-lab ISRAEL 2% Gibco US A. 0.5% Scientific Protein Labor Inc. TABLE V (continued) Peptone ap in medium __ U / ml Tryptose phosphate broth 0.5% Difco Prematone K 0.5% Humko Sheffield Chemicals. cells (105 cells / 30 mm dish) are placed in medium with 10% HS, and 48 hours later

  medium is replaced by fresh medium containing the pep-

  indicated tone The amount of a p in the medium is determined

mined after 24 hours.

Claims (8)

claims   1 process for producing plasminogen activator (a.p) by cultivation, characterized in that a strain of diploid fibroblasts, fixed on surfaces, is cultivated   coated or uncoated, by a number of   in a culture medium containing serum, and then cultured the cells thus conditioned in a culture medium suitable for the production of p, from which it is collected at p. Process according to Claim 1, characterized   that diploid fibroblast cells are   IMR-90, HEL-299 or WI-38 cells.   Process according to claim 1 or 2, characterized in that the cells are grown on the surfaces   not coated with plates, leaves, grains, or others.   4 Process according to one of claims 1 to 3, characterized   in that the cells are conditioned by culture.   on surfaces coated with poly-D-lysine, protamine sulphate or gelatin, or on a cell matrix, and are subsequently cultured   on a coated or uncoated surface.   Method according to one of claims 1 to 4,   in that the cells are conditioned in a   medium containing horse serum (HS) for 3 to 12 -   days, then grown in a medium containing lactalbumin drolysate (LH).   Process according to one of claims 1 to 5,   in that the cells are conditioned for   A p culture is obtained in a medium containing fetal calf serum (FCS), pig serum, human cord serum, newborn serum, dog serum, serum of fetal calf serum.   goat, or chicken serum, and are subsequently   in a medium containing a suitable hydrolyzate for produce the desired.   Process according to Claim 6, characterized in that the production of α p is carried out by cultivation in   a medium containing a peptone selected from hydro-   casein lysate, tryptose phosphate broth pep-   Tone D M, Prematone-K, or other equivalent product of the trade.   Process according to one of claims 1 to 6,   in that the product is removed continuously from perfusion culture.   Process according to one of claims 1 to 8,   characterized in that a p is adsorbed on silica, released   by contact with a base and purified by conventional means. Siques.   Method according to one of claims 1 to 9,   characterized in that the medium is recycled after removal of a p.   11 Plasminogen activator obtained by the se- one of claims 1 to 10.