JPH0137115B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF THE INVENTION The present invention relates to a method for producing plasminogen activator (pa) by a special method of culturing certain cells. The present invention provides novel culture methods and certain cell lines that exhibit high PA productivity without transformation with viruses or treatment with carcinogens or co-carcinogen compounds (inducers). The cells have unchanged chromosomal and growth characteristics at the end of the production cycle and can be grown and recycled under standard conditions used for each cell line. BACKGROUND OF THE INVENTION Urokinase is a proteolytic enzyme used as a component of therapeutic agents for blood clots. Its efficacy has already been proven and its use is permitted according to US FDA regulations, as well as in Europe and Japan. The two enzymes called urokinase and plasminogen activator are thought to be essentially the same. Currently, urokinase is mainly produced from urine, but it exists in urine in extremely small amounts, at only 8 units/ml. The cost of preparing the final product is very high and it is only available in small quantities. Therefore, efforts are being made to develop methods to produce PA on a commercial scale. SUMMARY OF THE INVENTION The present invention relates to a method for producing a plasminogen activator (hereinafter referred to as pa) and the enzyme produced. The present invention is based on the unexpected finding that the enzyme pa can be produced on a commercial scale from common diploid cells. As explained in detail below, cells capable of producing pa were isolated and adjusted to produce a large amount of pa. Certain cell lines of established human diploid cells, such as IMR-90, WI-38 or HEL-299, are first cultured in a medium containing serum other than fetal calf serum, preferably horse serum, for about 5-5 hours. It has been found that when cultured for 15 days or on a plate coated with a matrix such as poly-D-lysine, it acquires the ability to produce pa. The thus prepared cells continuously produce and secrete pa for at least 15 days when cultured in a serum-free medium containing lactalbumin hydrolyzate or other suitable nutrient medium.
When pa accumulates in the medium and reaches a certain concentration,
Pa production decreases and eventually stops. Maximum concentrations are approximately 1 unit/ml for many common cell lines, 3-5 units/ml for transformed cells, and 30 units/ml for pig bladder cells.
-50 units/ml, IMR-90 is 100-300 units/ml. Continuous removal of PA from the nutrient medium avoids reaching a maximum concentration and the cells produce PA for a significantly longer period of time. In IMF-90, pa is produced at a rate of approximately 50 units/day/ ¹⁰⁵ cells. pa is also recovered from the medium and recycled. PA produced by fibroblasts was found to have a molecular weight of 65-75 KD. It reacted with polyclonal and monoclonal antibodies raised against TPA. IMR-90 is a cell line similar to WI-38, which is approved as a source of live vaccines for polio and rubella. The cultivation of these cell lines has been described in detail in the literature, and the optimal conditions are well known. The method according to the present invention will be explained using the IMR-90 cell line as an example. This is just an example;
It should be understood that new cell lines that are similarly established or have similar properties can be used as well. Furthermore, the following description should also be understood as merely an example. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to the production of plasminogen activator (pa), which comprises culturing certain diploid cells under specific conditions and recovering the target product from the culture. The preferred cell line used is IMR-90, which was described by WW Nichols (1977) at 16
A human diploid fibroblast cell line obtained from the lungs of a week-old female fetus. The cells are ATCC (CCL186, 7th
obtained from subculture). IMR−90, WI−38,
There is no report that HEL-299 produces significant amounts of pa. In this study, they were initially considered to be non-producing strains, but by culturing them in medium containing horse serum and then in other nutrient media, we were able to convert them into significant producers of pa. I found out what I can do. I solved the IMR-90 obtained from ATCC,
Planted in 10% FCS-containing medium recommended by ATCC. After one passage culture, cells were transferred to LH medium and enzyme content was measured. As a result, slight production of pa (less than 5 units/ml after 4 days) was observed. After two passages in FCS, IMR-90 cells were grown at 10% HS.
Secondary culture was performed in the containing medium. After four passages (approximately 12 divisions) in this medium, cells were transferred to LH. The first
The results in the figure show that the cells became a very high pa producing strain. The enzyme is rapidly produced;
The concentration in the solution reached approximately 100 units/ml. The same cells were then subcultured continuously in HS,
I looked into pa production from time to time. The results in Table 1 indicate that the ability to produce pa was maintained until the cells began to show signs of senescence, ie, slow division and flattening. This seems to correspond to the 65-70 split22-24
It was also observed after passage.

【table】

[Table] Cells were continuously subcultured in HS medium according to standard procedures. To measure pa production, cells were plated into HS medium after trypsin digestion (2×
¹⁰⁵ cells, 30 mm plate, 1.5 ml medium), and after 24 hours the medium was changed to LH. Activity was measured in aliquots of the culture medium 96 hours after LH transplantation. Blank tests performed on plates without plasminogen were negative. Thaw frozen IMR-90 after 7th passage
2 passages (8th and 9th passages) in DMEM/FCS
Cultured. Initial experiments were performed with cells frozen after the 9th passage and stored in liquid nitrogen. unravel the cells,
It was subcultured for 2 passages in DMEM/FCS and then in DMEM/HS. * Passage number assigned from the 8th passage. ** 0.5×10 ⁵ cells To exclude the possibility of pa production due to special non-reproductive mutations, the experiment was repeated from cells frozen after the 9th passage. As a result, it was found that the PA production capacity could be reproduced. Under certain conditions, the fibroblast cell line can be conditioned to produce pa by culturing on certain substrates in the presence of serum. Cells were grown in various types of serum using culture vessels previously coated with the various compounds listed below, and the expression of PA production ability was monitored. The amount of pa in the medium was found to vary depending on both serum and matrix.
The results obtained with poly-D-lysine coated vessels (Table 4) indicate that cells can adapt to produce pa on this matrix even in media such as FCS, which do not normally contribute to enzyme production. It shows.

Table: Once adapted, these cells continued to produce enzymes in serum-free medium as described above. The regulatory cells are used as means of production. According to the above observations, the production was specified in some detail. In these experiments, cells were grown in HS-containing medium, transferred to LH-containing medium, and the amount of pa in the medium was followed. The results summarized in Figure 2 show that for cells at early passages, the upper limit of enzyme concentration in the medium was reached at about 100 units/ml. This upper limit increases in later passage cells. In very old cells, enzyme production did not stop even when the enzyme concentration in the medium reached 300 units/ml. These results strongly suggest that the amount of PA in the medium is determined by PA formation and some negative factors. Negative factors include inhibitory feedback on enzyme synthesis or secretion or the production of other cellular products that inhibit the enzyme or catalyze its breakdown. Repeated passage of cells in HS-containing medium is thought to reduce this negative repression, resulting in enhanced high enzyme activity in the medium. Since 100 units/ml of activity corresponds to an enzyme concentration of approximately 1 mg/ml, the production amount in the above experiment was 1-3 mg/ml.
This is the percentage of Harvesting and Purification PA produced by cells can be harvested by adsorption from the culture using adsorbents known to adsorb urokinase, by concentration using membranes, or by a combination of both. Two methods are illustrated below. 1 LH medium containing adsorbed pa was treated with pulverized silica.
That is, add 1-2 mg of solid per ml of solution,
Mixed for an hour. The solids were then collected by centrifugation. The supernatant contained less than 5% of the pa originally present and was reused as described below. As a result of solid analysis, pa is quantitatively liberated from silica upon treatment with bases, especially ammonia, organic amines, and polymerized amines, and the resulting pa solution is further purified by various methods used in this field. I was able to do that. 2. Dialysis concentration The LH medium containing pa was transferred to a dialysis bag, and a water-absorbing substance was placed on the outside. The enzyme was concentrated 10-20 times with a total recovery of over 75%. Effect of medium replacement on pa Considering the similarities between the time course of pa production by IMR-90 and the time course previously observed in other cells, we investigated the effect of repeated medium replacement. As seen previously, as shown in Figure 2, when the enzyme concentration increases during the cultivation of IMR-90, the enzyme production rate decreases, but this slowdown is largely eliminated by irrigation.
Irrigation clearly interferes with the negative feedback regulation mechanism described above. The results in Figure 3 show that medium replacement affects pa production when a significant amount of enzyme has accumulated in the medium, but does not affect the initial production of pa. This was demonstrated by an experiment (Figure 4) in which cells grown in FCS medium were transferred to LH. These cells produced pa so slowly that its concentration in the medium was low, and pa production was not affected by medium replacement. Since the cost of the medium appears to be an important economic factor, experiments were conducted to investigate the possibility of medium circulation. The used medium was treated with silica to remove pa and diluted 1:1 with fresh medium back. It was found that more than 90% of PA was removed from the medium by silica treatment. The results shown in Figure 5 show that the culture medium can be used repeatedly. In fact, it was observed that higher amounts of pa were produced using circulated media than when fresh media was irrigated. The medium used for pa production in the above experiment was pa
Contains a hydrolyzate of lactalbumin, similar to that commonly used in related experiments. However, even if various peptones, which are hydrolysates of animal and plant-derived proteins and are used as substrates for microbial growth, are used,
It was found that pa was produced. An example of the results is shown in Table 5.

[Table] Cells were plated at 10 ⁵ cells/30 mm dish in a medium supplemented with 10% HS, and after 48 hours, the medium was replaced with a fresh medium containing the indicated peptone. p. in the medium.
a. was analyzed 24 hours later. Lactalbumin hydrolyzate 0.5% bio・
Bio-lab, Israel Casein Hydrolyzate 2% Gibco, USA DM Peptone 0.5% Scientific
Scientific Protein
Labor.Inc.) Tryptose phosphate broth 0.5% Difco Prematon-K 0.5% Humko Sheffield Chemicals Culture of IMR-90 cells 1 Standard procedure Cells were cultured in a conventional manner using Falcon.
DMEM and F using a 90mm plastic plate
-12 in a 1:1 (volume ratio) mixed medium,
Fetal calf serum or horse serum from Gibco USA was added to a concentration of 10%. Each plate was seeded with 5×10 ⁵ cells and 7 ml of medium. Confluence was reached on days 3-5 with approximately 8 x ¹⁰⁶ cells per plate. Cells were tryptic digested with 0.25% trypsin solution (Biolab, Jerusalem) for subculture. Trypsin solution (2-3 ml) was added to the plate and after 1 minute at room temperature most of the trypsin solution was sucked out, leaving the cells covered with a thin film of solution. After 15-20 minutes, a small amount of fresh medium was added and mixed with the cells using a pipette several times to obtain a cell suspension. Fresh medium (5 ml) was then added to each plate and a portion was removed for cell counting and re-implantation. Cells were also cultured using other plate sizes or other cell culture vessels at the surface area to cell ratios described above and following the same general procedure. 2 Effect of Substrate on IMR-90 Attachment and Growth Culture of IMR-90 on various substrates and coatings was investigated. To obtain a combined effect on engraftment efficiency and proliferation, cells were plated and grown on various substrates for 4 days. The results in Tables 2 and 3 show that the culture can be improved by coating the plates. The simplest and most effective coating is gelatin. moreover,
Gelatin coatings also allow growth on other surfaces such as Gelbond.

[Table] Experiments were performed from 1×10 ⁵ cells using 1.5 ml HS medium on a 30 mm plate. For experiments using Gelbond, a circular film was made to cover the bottom of the plate. Gelatin was used as a 0.03% aqueous solution and was left on the plate for 20 minutes and removed by aspiration. After 96 hours of growth, cells were separated by trypsin digestion and cell numbers were counted. * Gelbond is a product name for polyester thin film (marine biocaloids,
Marin Biocaloids). One side of the membrane is hydrophilic, and that side was used for the experiment.
It is not clear what method was used to make it hydrophilic, but it seems likely that an alkali was used.

[Table] The experimental details were the same as in Table 2, except that different cell groups were used and 2.6 x 10 ⁴ cells were plated in 30 mm plates. Plates were treated with protamine sulfate or poly-D-lysine (0.1 mg/ml). After incubating for 5 minutes, the plates were washed twice with sterile water and once with medium, and the cells were transferred to the plates. The cell matrix was made by growing a monolayer of CCL6 cells (human intestine, epithelium) and using 0.5% Triton X-100-
Adjusted by treatment with water. After incubating for 10 minutes, the plates were washed twice with PBS and once with medium. At this stage, the plate is ready for cell implantation. Units Enzyme activity is expressed in CTA units. Enzyme analysis was performed using the fibrin plate method assayed with standard urokinase obtained from the NIH (USA).
This was done using the plate method. Characteristics of cells after use for PA production Karyotype analysis of IMR-90 adapted for PA production as described above was performed. Normal complement of chromosomes (2N
=46) showed the same experimental results as reported for the parental cell line. Cells used for pa production in LH medium for 12 days were transferred to growth medium containing 10% FCS and grown under the standard conditions described above. When the cells were followed for one month, the growth rate, cell concentration at confluence, and morphological appearance of the cells were the same as the parent IMR-90. PA production was also below normal cell levels. Since similar results were obtained with cells of cell lines such as WI-38 and HEL-299, the present invention also extends to the production of pa by these cell lines.

[Brief explanation of drawings]

Figure 1 shows the production of pa by IMR-90 cells grown in HS medium. Cells were grown for 4 passages after passage 8 in horse serum (HS) containing medium. Second
The figure shows IMR incubated in DMEM/HS for various times.
-90 shows the change in pa production over time. passage number
12 (set-1), passage number 15 (set-1), passage number 20 (set-1), passage number 14 (set-2),
Passage number 20 (set-2). Figure 3 shows the effect of medium replacement on pa production by IMR-90 cells grown in HS medium. IMR-90 cells after passage 15 (passage 6 in HS) were plated at 1×10 ⁵ into 60 mm Petri dishes containing 2.5 ml of HS medium. After 24 hours, the medium was changed to LH. At the time indicated by the arrow,
Take half or all of the medium from each plate,
Replaced with fresh LH medium. 100μ in control plates
The medium was taken for pa analysis and replaced with fresh LH medium. 〇 means complete medium replacement, △ means half medium replacement,
● indicates medium-free replacement. Figure 4 shows the effect of medium replacement on pa production by IMR-90 cells grown in FCS medium. IMR-9 after 9 passages in FCS
Cells were plated (5×10 ⁴ ) into 16.4 mm wells containing 2 ml of FCS-containing medium. After 24 hours, remove the medium.
The experiment was continued by replacing it with one containing LH. ○ indicates total medium replacement, △ indicates half medium replacement, and ● indicates no medium replacement. Figure 5 shows continuous production of pa by IMR-90 cells. IMR after 23 passages (13 passages in HS medium)
−90 cells in 60 mm containing 2 x 10 ⁵ HS-containing medium 2.5 ml
The plate was transferred to a Petri dish. 24 hours later medium
It was changed to LH, and then changed to the time indicated by the arrow.
In group 1, half of the medium from each plate was collected;
Replaced with fresh LH medium. In group 2, the entire medium was collected. 1.25 ml of fresh LH medium was added to the cells. The collected medium was treated with silica to adsorb pa, and 1.25 ml of this medium was returned to each plate in group 2. pa activity was measured in a portion of the medium at each time point. ● indicates group 1, ○ indicates group 2.

Claims (1)

[Scope of Claims] 1. For multiple passage culture in a medium containing horse serum, newborn calf serum, dog serum, goat serum or young chicken serum,
Diploid fibroblast cell lines were cultured for 3-12 days on surfaces coated with D-lysine, gelatin or cell matrices, then treated with casein hydrolyzate, DM peptone tryptose phosphate broth, prematon-
A process for producing pa, characterized in that the conditioned cells are cultured in a peptone-containing medium selected from K or other commercially available equivalents to obtain plasminogen activators (pa), which are harvested from the medium. 2. The method according to claim 1, wherein the produced pa is continuously removed from the culture by perfusion. 3. The method according to claim 1 or 2, wherein 3pa is adsorbed onto silica, brought into contact with a base to be removed, and then purified by a conventional method. 4. The method according to claim 1, wherein the medium is recirculated after removal of pa.