DD251995A5 - Process for the preparation of tissue plasminogen activators - Google Patents

Abstract

The invention relates to the production of tissue plasminogen activators using novel serum-independent human cell lines, proteins are suitable in appropriate pharmaceutical form for the prophylactic and therapeutic treatment of the human body. The aim of the invention is to make the method economical. The object of the invention is the provision of a new production method with the aid of a human cell line which operates independently of serum. According to the invention, the method consists in culturing a serum-independent cell line in a serum-free medium and isolating the tissue plasminogen activators from the harvest fluid.

Description

Field of application of the invention

The invention relates to the production of tissue biotin activators by means of human cell lines that proliferate in the absence of serum or any exogenous macromolecular growth factor. The proteins produced by said cells can be used in the treatment of certain human diseases.

Well-known technical solutions

With the recent development of recombinant DNA technology and the development of cell culture techniques, the possibility of controlled biological production of useful and pharmacologically interesting compounds - especially proteins - such as interferon, insulin and antigens has emerged. There is an increasing demand for the development of biological systems that ensure the large-scale production of other proteins of biological and especially pharmacological interest.

The term "protein" as used above as well as hereinafter is meant to include high molecular weight polypeptides - e.g., above about 34,000 - but also lower molecular weight polypeptides - eg, below about 34,000 - and their derivatives, such as glycosylated, phosphated and sulfated derivatives.

- λ - ^ OI 33Ο

Advances in the field of recombinant DNA technology make it possible to introduce a gene encoding a desired protein into microorganisms and then cause the microorganisms to synthesize the protein. However, many biologically important molecules can not be synthesized by this technology. This applies in particular to those molecules whose structure is not yet known. In most cases, the proteins secreted by genetically modified microorganisms are not a faithful reproduction of the authentic molecules, but differ from the latter in the N and C terminals of the amino acid sequence. This fact is due to the experimental procedure in recombinant DNA technology. In addition, glycosylated proteins can not be produced by microorganisms such as bacteria and, to some extent, yeasts lacking the requisite cellular mechanism. In many cases, use can be made of the cell and tissue culture technique more advantageous. Since cell cultures are derived from intact organisms, the proteins produced by the cell cultures correspond to the naturally occurring proteins in all respects.

Nonetheless, the cultivation of higher organism cells - such as mammalian cells - on a large scale is a difficult problem. The nutrient requirements of such cells are more stringent than those of most microorganisms that grow in artificial media. The growth medium of most of the hereditary cells described so far includes more expensive serum. The cost of serum greatly determines the economic feasibility of the cell culture technique and may limit its applicability to the production of otherwise unavailable proteins. Some cells can be cultured in serum-free medium supplemented with hormones or growth factors such as transferrin, insulin, epidermal, fibroblast or nerve growth factor. In most cases, the cells will not proliferate indefinitely in these serum-free media.

Cell lines growing in a serum-free medium are of particular importance in the production of those proteins susceptible to destruction or contamination by serum or exogenously supplied growth factors. Such a protein is, for example, pro-tissue plasminogen activator (Pro-TPA).

The so-called plasminogen activators have been the subject of scientific research demonstrating their evident clinical applicability in dissolving blood clots. Blood clots are composed of fibrin, which has been formed from its soluble precursor fibrinogen under the action of the enzyme thrombin. They are a major cause of illness and mortality in humans; their resolution without side effects is difficult to achieve. Mammalian plasma contains an enzymatic system capable of dissolving the fibrin in blood clots. One component of the fibrinolytic system consists of the enzymes - plasminogen activators - which converts plasminogen (an inactive proenzyme form of plasmin) into the proteolytic enzyme plasmin. Plasmin then breaks down the fibrin network of the blood clots to form soluble products. In cases where the thrombolytic potential of the body is insufficient to remove formed intravascular thrombi, for example in patients suffering from thromboembolism or postoperative complications, it may prove necessary to administer additional thrombolytic agents. There are two activators of human plasminogen that are commercially available for thrombolytic therapy: urokinase, a serine protease isolated from human urine or human cultured kidney cells, and streptokinase, a streptococcal-recoverable bacterial protein. Since neither of the two enzymes has a specific affinity for fibrin, thrombolysis with these substances is associated with a systemic activation of plasminogen, which can cause indiscriminate processing of coagulation proteins and thus a significant increase in the risk of internal bleeding (hemorrhage) during treatment. Another disadvantage of urokinase is its very short useful half-life after its injection in humans. For this reason, high urokinase doses are required to achieve effective fibrinolysis. Streptokinase is a protein foreign to humans, which in turn creates the risk of neutralizing antibodies that block its action and lead to allergic reactions that can be painful and potentially fatal.

Another group of plasminogen activators, called "tissue plasminogen activators" (hereinafter referred to as "TPAs"), are known to exist in most human tissues, TPAs derived from different tissues may be different in molecular properties, immunological They differ from urokinase in their chemical and immunological properties, in terms of their greatly enhanced fibrinolytic activity in the presence of fibrin, as well as their high affinity for fibrin (see S. Thorsen et al., Thromb Haemorrh., 28, 65-74 [1972], DC Rijken and D. Collen, J. Biol. Chem., 256, 7035-7041 [1981]). Because of their high affinity for fibrin, the effect of TPAs is limited to that Blood clots themselves, which significantly reduces the risk of uncontrolled bleeding shows the relationship between plasminogen, plasmin, fibrin and the various plasminogen activators.

UROKIMSB STREPTOKINASE

Plasminogen (Inactive progenitor in the blood)

plasmin

(Blood-active enzyme that dissolves blood clot and activates Pro-TPA

TISSUE PLASMINOGEIACT IVAT OR

4 \

PRO-TISSUE-. '. ,

PLASMINOGEIAKTIVATOR

Recently, two patients suffering from coagulation failure have been successfully treated with a TPA isolated from the culture fluid of a human melanoma cell line (see W. Weimar et al., The Lancet [1981], 1018-1020). There are two molecular forms of TPA: the active two-chain form and an inactive one-chain form (precursor TPA or "Pro-TPA"; see also DC Rijken and D. Collen, loc cit, DC Rijken et al., J Biol. Chem. 257, 2920-2925 [1982] and P. Wallen et al., Progr. Fibrin., 5, 16-23 [1982]). Pro-TPA can be converted into the active TPA by incubation with fibrin or by the Influence of plasmin are converted, which triggers its own synthesis by this cascade-like reaction.

Human TPA sources include extracts of various human tissues (which are not available for commercial use) as well as various human tumor cells that have been shown to release TPAs to varying degrees (EL Wilson et al. , Cancer Research 40, 933-938 [1980]; EL Wilson et al., Blood 61, 568-574 [1983]).

EP-OS 41,766 (inventors D. Collen, DC Rijken and O. Matsuo) describes a 72,000 molecular weight TPA isolated from a cultured human melanoma cell line (Bowes) and which is likely to be linked to the TPA, which has already been described by EL Wilson et al. (Cancer Research 40, 933-938 [1980]). Like other cell lines already known, this human melanoma cell line - Bowes - requires the presence of serum such as fetal calf serum for its growth. However, serum is very expensive and also contains proteinaceous components which contaminate the produced TPA and prevent the isolation of large amounts of Pro-TPA. This can lead to a tedious and laborious cleaning procedure on either TPA or Pro-TPA.

Object of the invention

The present invention overcomes the disadvantages of the known processes for producing TPA and enables economically efficient production of TPA with the aid of new human cell lines growing in the absence of serum or any exogenous macromolecular growth factor.

Essence of the invention

The object of the invention is to develop a process for the production of TPA with the aid of a serum-independent human cell line or a serum-independent mutant cell line derived therefrom.

According to the invention, new human cell lines are used which grow in the absence of serum or any exogenous mollomolecular growth factor such as insulin, transferrin, epidermal growth factor, etc. The cells used in the invention are capable of adhering to the culture vessel even in the absence of serum or fibronectin.

The main advantages of the novel cell lines used according to the invention are:

a tremendous cost saving, since serum such as fetal calf serum is very expensive, the extremely cheap and easy purification of the products produced by the new cell lines, as it is. There is no serum contamination in the medium and the product can be readily purified from the serum-free medium, and the possibility of producing proteins that can not be recovered (or only in low yields) from serum-dependent cell lines due to the presence of proteases in serum ,

The process of making the new serum-independent human cell lines consists of the following steps:

a) removing serum-containing medium from the culture of a serum-dependent human cell line and replacing it with serum-free medium,

b) delivery of serum-free medium to the coalesced or non-coalesced cells, and

c) allowing the cells to grow until a serum-independent cell line has formed.

The term "serum-free medium" as used hereinbefore and hereinafter is intended to mean a medium to which not only serum but also any macro-dermocular growth factor has been withdrawn Accordingly, a cell line obtained in the absence of both serum and serum exogenous macromolecular growth factors grows, referred to as "serum-independent cell line".

The serum-dependent human cell line used in the above method as a "starting material" is specifically a continuous line and can be derived from human neoplasms such as melanoma, tetratom, sarcoma, glioblastoma, meningioma, neuroblastoma, lipoma, adenoma, breast or Preferred cell lines are those that produce TPA and / or pro-TPA In a preferred embodiment of the present invention, a human melanoma cell line, specifically the Bowes melanoma cell line (hereinafter referred to as "Bowes I" cell line referred to) used. Cell lines derived from normal human tissue may also be used as "starting material", but with limited in vitro lifetime (growth ceases after, for example, about 50 cell divisions) and are of lesser importance with respect to the present invention.

The serum-free nutrient medium is, for example, a commercially available medium such as minimal Eagle's medium (MEM), MEM spinner medium, Dubecco's modified Eagle's medium (DMEM) or Roswell Perk Memorial Institute culture medium (RPMi) -1640. Likewise, other equivalent media may be used. The serum-free medium may contain a sufficient amount of a buffer, such as sodium bicarbonate, to maintain a stable p-value. Since cultured cells of the complete immune defense system are deprived as an integral part of the intact organism, antibiotics such as penicillin, streptomycin, tylocine and the like are advantageously included to prevent infection of the culture.

Normal human cells will grow properly only when attached to the surface, whereas tumor cells often thrive better when in suspension. Laboratory vessels for anchoring dependent cells are well known in the art. They range from "microwave" bowls and flat-bottomed petri dishes to tissue flasks of various sizes or cylindrical bottles, referred to as roller bottles that rotate uninterrupted. "Anchor-independent cells can be grown in culture vessels that are moved mechanically or by mixing with a gas stream The process can be carried out, for example, in a tissue culture flask with a coalesced or nearly coalesced monolayer of a human cell line such as a human melanoma cell line - especially Melanoma Bowes I grown in a serum-containing nutrient medium is carried out in a humid atmosphere containing carbon dioxide, for example in a humid atmosphere of about 95% air and about 5% CO ₂ , at a temperature between about 35 ⁰ C and about 40 ⁰ C and in particular at about 37 ⁰ C. D The serum-containing medium is discarded and replaced with a serum-free medium such as RPMI-1640 or DMEM. After a few days, the cells begin to detach from the piston surface and float freely in the medium. After a few weeks, most of the cells will have died in a serum-free or growth-factor-free medium. To maintain the viability of the cells, a conditioned medium is added to the cultures during the adjustment period to serum-free medium. The conditioned medium may be collected from a companion cell culture which has been kept in serum-free medium for a period of time, for example 24 hours. Serum-free medium containing conditioned medium must be changed at intervals of, for example, 3 to 7 days until the number of cells has increased sufficiently, ie until about one third of the surface of the vessel is covered. At this stage, the conditioned medium is no longer needed and the cells are now fed exclusively with serum-free medium. After a confluent monolayer has formed, the cells must be passed through, for example, vigorous regurgitation of the vessel, removing most of the cells from the surface, or with the aid of ethylenediaminetetraacetic acid (EDTA) in order to ensure the survival, the fresh culture of the fresh culture is carried out at a sufficiently high cell density. Culturing and passing the cells at confluence is continued until the cells grow as stable and adherent monolayers. In this way, a serum-independent cell line is established over a period of, for example, 1 to 6 months, especially 1 to 3 months.

In an alternative variant, the serum-independent cell line can be applied without the addition of conditioned medium collected from the serum-dependent companion cell culture, as long as the cell density is high enough. In this case, the cells condition their own medium. The cell density can be kept high by not excreting the non-adherent cells. After a few days in the serum-free medium, for example, the majority of the cells detach from the tissue culture vessel and float freely in the medium described above. The peeling cells remain viable as long as they are maintained at a sufficiently high density. When the serum-free medium containing the non-adherent but viable cells is centrifuged and the cell pellet is taken up in fresh serum-free medium and returned to the flask containing the few adherent cells, the adherent cells will begin to grow. In this case, the cells in suspension together with the adherent cells condition their own medium. After two to three weeks, the cells previously in suspension will begin to re-attach themselves to the surface of the culture vessel, these cells will adhere with time and, once re-grown by vigorous regurgitation or with the aid of EDTA be removed again as described above. Another method of preparing the "conditioned medium" in the presence of the adherent cells is to replace a portion of the serum-free medium - approximately half of it - with fresh serum-free medium and repeat this procedure every 24 to 72 hours until a sufficiently high cell density is achieved The cells may then be passed (for example, using EDTA) At the first passage, the fresh serum-free medium is about 40% of the conditioned medium removed from the culture prior to EDTA treatment The cell density should be sufficiently high, ie corresponding to about 30% or higher - preferably about 60 to 70% - confluency at about 20% confluence or at about 1 x 10 ⁵ cells / ml in the case of suspension cultures, di e cells conditioned medium for their growth. At a high cell density, ie at about 60 to 70% confluency or at about 2 x 10 ⁵ to 5 x 10 ⁵ or more cells / ml in the case of suspension cultures, the cells will grow and proliferate in the serum-free medium without the addition of conditioned medium ,

The serum-independent cell lines can be used as adherent cultures - for example on the inner wall of a laboratory vessel - or as suspension cultures.

It is also possible to use mutant cells which can be obtained from the cells according to the invention and which are able to proliferate in a serum-free culture medium The mutants form spontaneously or can be prepared in a manner known per se. The mutants can be obtained, for example, by chemical means such as by the action of a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine or mustard oils, or by irradiation with, for example, ultraviolet or X-rays.

Preferred serum-independent cell lines and mutant cell lines used in accordance with the invention are those which produce TPA and / or pro-TPA.

The human cell lines used in this invention, as well as the mutant cell lines derived therefrom, have an indefinite lifetime and can be used in a similar manner to the original serum dependent cell lines; such as for the commercial production of biologically active compounds such as proteins such as interferon antigens, angiogenic factors and in particular plasminogen activators under serum-free conditions or for the production of hybridoma cell lines.

As already mentioned above, the melanoma cell line Bowes I is known to excrete tissue plasminogen activator to a high degree (see EP 41766); in the above process, it is preferably used as the "starting material." The resulting serum-independent new cell line is hereinafter referred to as "Bowes II".

The new serum-independent cell lines differ from the corresponding serum-dependent parent cell lines in many respects. For example, Bowes II cells differ from the parental Bowes I melanoma cells in their morphological appearance. Examination under the phase control microscope shows that Bowes-Il cells show markedly more frequent mitoses compared to serum-spilled Bowes I cells. The addition of serum to Bowes II cells causes a striking morphological change.

The preferred serum-independent cell lines of the present invention - especially the Bowes II cell line - excrete highly tissue piasminogen activates which, unlike the parental serum-dependent cell lines such as the Bowes I cell line, are predominantly proactivators. Apart from TPA and Pro-TPA, the new lines also produce other pharmacologically valuable substances. For example, the Bowes II cell line produces its own growth factor as well as a tumor neurotransmitter.

Culturing serum-independent human cells and harvesting the culture fluids Serum-independent human cells can be cultured substantially in the manner described above. For example, Bowes II cells are seeded in tissue culture flasks of sufficient cell density to ensure survival, and stored in a serum-free medium - e.g. B. RPMI-1640 -, supplemented with antibiotics, at a temperature between about 35 ° C and about 40 ° C - especially at about 37 ° C - grown in a humid air atmosphere containing about 5% CO ₂ . The harvesting of harvesting liquids can be started as soon as the cells become adherent, and it can be repeated, for example, at 23-hour intervals. The daily harvesting of harvest fluids can be continued until the monolayer coalescence requires transfer to fresh culture vessels. Since the proliferation rate of Bowes II cells cultured in serum-free medium is very low (doubling lasts up to 6 days, compared to a doubling time of about 1 day in the Bowel I parental melanoma cells), the transformation is only at longer intervals (at intervals of about 4 to 6 weeks), and the harvest liquid can be collected from a flask for a period of about 3 to 6 months, thereby saving considerable time and effort, and collecting the liquids to a simple one Task is done. Likewise, it is possible to increase the proliferation rate of Bowes II cells by occasionally adding a growth factor, such as. B. insulin is added. Petri dishes, tissue culture flasks, and other laboratory useful vessels are insufficient to provide a sufficient surface to volume ratio for the large scale, practical culture of surface-adherent serum-independent cells. For large-scale production, the area-volume ratio can be increased by various tools known in the art. For example, the cells can be grown on spongy polymers, on slices of thin slices, on small pellets, and the like. Alternatively, the new serum-independent cell lines, such as Bowes II, can be grown in suspension. Large scale fermentation taverns suitable for suspension culture are well known in the art; Si ₁ B were developed by modifying the fermentation tanks for unicellular microorganisms. In order to keep the cells evenly suspended in Mediurp and evenly distribute dissolved gases (eg oxygen), agitation is required. This can be done by conventional turbine stirrers, propeller stirrers, vertically oscillating vibratory mixers, and the like. A slow stirring is preferred in order to protect the cells from damage. Homogeneous agitation can also be maintained by passing a gas stream through the culture.

The harvested fluids from serum-independent human cells, such as Bowes II cells, must be centrifuged to remove detached cells and cell debris. It is advisable to add a non-ionic detergent to the medium so as to prevent the adsorption of TPA, Pro-TPA and other valuable substances secreted by the cells on the surface of the vessel and to preserve the enzyme activity. Preferably, the nonionic detergent such as Triton X-100® or Tween 80® is added at a final concentration of about 0.01 to 0.1%. Since the highest activity is maintained at a pH of 5.5 to 6.0, the harvest fluids are acidified with a weak acid. This is done for example with a lower alkanoic acid such as acetic acid, the liquids before the low temperature storage -. B. at about 4 ⁰ C for a 48-hour storage or at -20 ⁰ C for extended storage - be adjusted to said pH.

The content of TPA, Pro-TPA and other valuable substances in the harvest fluids can be determined using conventional techniques. For example, the TPA content can be measured using the ¹²⁵ L fibrin assay or by a fluorometric assay method. In the former method, the time course of plasminogen-dependent release of radioactive fibrin degradation peptides from ¹²⁶ L-labeled fibrin was measured, which was applied as an insoluble coating on the surface of plastic racks (see EL Wilson and E. Dowdle, Int 22, 390-399 [1978]). In the second case, the fluorescence increase resulting from the amidolysis of a suitable synthetic substrate such as Cbz-Gly-Gly-Arg-AMC (AMC: aminomethylcoumarin residue) or Boc-Val-Gly-Arg-AMC is measured (see M Zimmermann et al., Proc Natl Acad., USA 75, 750-753 [1978]). While the ^'25 I-fibrin assay indicates the total enzyme activity of both TPA and Pro-TPA, the iluorimetric determination can be made in a manner that measures only TPA activity or, alternatively, that after

Treating the harvesting liquid with plasmin, which converts pro-TPA to TPA, both TPA and Pro-TPA are included.

Using the above TPA determinations it can be demonstrated that the harvestable harvestable liquids according to the invention contain Pro-TPA in a high percentage. This is probably due to the fact that the culture medium used lacks plasmin or other proteases which can convert pro-TPA to TPA. The preponderance of Pro-TPA in harvested fluids obtained, for example, from cultured Bowes II cells strikingly contrasts with harvest fluids from Bowes I melanoma cells that contained only a small amount of Pro-TPA.

Isolation and Purification of Proteins Secreted by Serum-Independent Cells The isolation of the desired proteins from the culture fluid obtained from serum-independent cells such as Bowes II cells and their purification can, in principle, be effected in any suitable manner as is customary in protein chemistry ; such as by fractional precipitation, for example, with inorganic salts, by gel filtration, for example, on crosslinked dextranes or agarose, by gel electrophoresis or by chromatographic means such as, for example, adsorption chromatography, ion exchange chromatography or affinity chromatography. Corresponding methods of isolation and purification of TPA and Pro-TPA include, for example, the following:

Chromatography on zinc chelated agarose, concanavalin A agarose and Sephadex G-150® (see D. C. Rijken and D. Collen, J. Biol. Chew 246, 7035-7041 [1981]). The harvest liquid is passed through a zinc chelator agarose column. The adsorbed enzyme is eluted with an imidazole-containing buffer. The solution thus obtained is applied to a concanavalin A agarose column. Elution can be done with methylmannoside and KSCN. In the final step, the enzyme is gel-filtered on a Sephadex G-150® column.

_K - Chromatography on Affi-Gel Blue® and Aminobenzamidine Sepharose® (see LC Gilbert and JT Wachsmann, Biochim Biophys Acta 704, 450-460 [1982]). The enzyme is sequentially adsorbed on Affi-Gel Blue® and Aminobenzymidine-Sepharose®. The desorption can be carried out with an arginine-containing buffer.

Selective adsorption of TPA and Pro-TPA on a carrier of specific affinity of soluble fibrin fragments covalently bound to an insoluble matrix such as dextran (see European Patent No. 23,860). The adsorbed enzyme can be eluted with an acetic acid buffer of pH 4.2.

Chromatography on an immunoaffinity column of anti-TPA antibodies - especially monoclonal anti-TPA antibodies - bound to an insoluble matrix such as Affi-Gel® or Sephadex-48®.

Affinity chromatography on DE-3-Sepharose®. The seeds of legume Erythrina latissirna contain a trypsin inhibitor designated DE-3, (FJ Joubert et al., Hoppe-Seyler's Zeitschr Physiol. Chem. 302, 531-538 [1981]). It has been shown that DE-3 is also capable of inhibiting TPA activity. Thus, the purified DE-3 inhibitor can be linked to an insoluble matrix using standard techniques. The medium containing TPA and Pro-TPA will adsorb and may be eluted by a chaotropic agent-containing buffer ₍ such as KSCN).

Electrophoresis on SDS (SDS-PAGE) containing polyacrylamide gels. This method is particularly suitable for analysis purposes as well as for the determination of relative molecular masses.

In order to obtain a sufficiently pure product, a single cleaning cycle or several successive purification steps can be selected. In addition, additional purification steps such as dialysis in a suitable buffer mixture, reverse phase HPLC, and the like may be required.

For example, the harvest fluids may be subjected to chromatography on zinc chelate agarose, concanavaiin A agarose, and Sephadex G-150® as a first isolation step (which may also serve to provide the desired protein present in the large volumes of harvest fluid then the enriched protein thus obtained can be finally purified by affinity chromatography.

In a preferred embodiment of the present invention, the harvested liquid obtained from serum-independent human cells such as Bowes II cells and containing TPA and Pro-TPA is centrifuged to remove whole cells and cell debris and is then passed through a column of room temperature at room temperature insoluble matrix to which an TPA and Pro-TPA selective affinity reagent is attached - for example, BrCN-activated Sepharose® - to which in turn the DE-3 inhibitor has been attached. After washing the column, the adsorbed TPA and Pro-TPA are treated by treating the column with a buffer solution such as phosphate buffered saline at pH about 5.5-6.0 and containing a chaotropic reagent such as KSCN.RTM. about 1.4 to 2.0 molar, preferably 1.6 molar - desorbed. Alternatively, benzamidine or arginine may be chosen as the desorbing agent.

In an alternative approach, TPA and Pro-TPA are isolated by chromatography on a monoclonal antibody column prepared by attaching monoclonal anti-TPA antibodies to, for example, Affi-Gel®. Advantageously, a detergent and especially a non-ionic detergent such as Triton-X-TOO® or Tween 80® is added to all of the buffer solutions used in the purification steps so as to prevent the adsorption of TPA and Pro-TPA on the vessel surface and the stability to improve. The detergent may be added to a final concentration of 0.01 to 0.1%.

The resulting purified solution contains TPA and Pro-TPA. For the production of TPA free of any Pro-TPA, the Pro-TPA can be enzymatically converted to TPA by, for example, the action of plasmin or an enzyme having an equivalent effect on Pro-TPA.

In a preferred embodiment of the present invention, Pro-TPA is isolated in substantially pure form and free of TPA: Pro-TPA is a true proenzyme, i. H. it is the enzymatically inactive form of TPA, which adsorbs Pro-TPA to a greater extent than TPA on fibrin, thus acting more selectively than TPA in inducing fibrinolysis since it is the first to attach to fibrin and only then to TPA, whereas in the case of TPA, there is limited possibility that it will activate some plasminogen in the bloodstream before that fibrin site where the localized effect is actually desired. For the production of substantially TPA for pro-TPA, advantageously during the purification process a protease inhibitor such as aprotinin or a basic pancreatic trypsin inhibitor is included to inhibit traces of proteases which may be present and which is a (partial) conversion of Pro-TPA in TPA can cause. The final purification is then carried out by chromatography on a column containing a selective affinity reagent, such as DE-3, in the presence of an inhibitor which, such as diisopropylfluorophosphate or nitrophenylguanidinobenzoate, selectively binds only TPA and not Pro-TPA. These reagents prevent TPA from reaching the

Affinity Säbs adsorbed. As a result, the bound TPA will pass through the DE-3 column, whereas Pro-TPA may adsorb to the column.

Accordingly, the present invention is to provide a process for the preparation of TPA, Pro-TPA or mixtures thereof which is characterized in that serum-independent human cells capable of producing TPA, Pro-TPA or mixtures thereof or their mutants such as Bowes II Cells are cultured in a serum-free medium and the desired proteins are isolated from the harvesting liquid and that - if desired - a mixture of the proteins thus obtained is separated or converted into the individual components. Specifically, the present invention provides a process for the preparation of Pro-TPA-free TPa, characterized in that in a recovered mixture of present Pro-TPA is enzymatically converted to TPA. Specifically, the present invention provides a process for the preparation of TPA-free pro-TPA which is characterized in that a protase inhibitor is included during isolation and that the purification, including the final purification, is carried out in the presence of an inhibitor which selectively inhibits TPA

 Pharmaceutical preparations

The novel proteins, especially TPA and Pro-TPA, which can be obtained according to the invention from, for example, cultivated Bowes II cells, have valuable pharmacological properties. Thus, TPA and Pro-TPA from Bowes II cells can be used in analogy to known plasminogen activators in humans for the prevention or treatment of thrombosis or other conditions where it is desired to have a local fibrinolytic or proteolytic via the mechanism of plasminogen activation To generate activity. This concerns diseases such as arteriosclerosis, myocardial and cerebral infarction, venous thrombosis, thromboembolism, postoperative thrombosis, thrombophlebitis and diabetes-related vascular diseases.

The invention further relates to pharmaceutical preparations containing a therapeutically effective amount of the active ingredient (in particular TPA, Pro-TPA or mixtures thereof) together with organic or inorganic, solid or liquid pharmaceutically acceptable excipients, the latter being for a parenteral, d. H. intramuscular, subcutaneous or intraperitoneal administration and must not develop adverse interaction with the drugs.

Particularly suitable appear infusion solutions and thereby preferably aqueous solutions or suspensions, it being possible to prepare them before use, for example, freeze-dried preparations containing the active ingredient alone or together with a carrier such as mannitol, lactose, glucose, albumin and the like. The pharmaceutical preparation can be sterilized and, if desired, mixed with additives such as preservatives, stabilizers, emulsifiers, dissolution aids, buffers and / or salts for regulating the osmotic pressure. Sterilization can be achieved by sterile filtration through small pore size filters (0.45 μπ \ diameter or smaller), after which the preparation can be lyophilized if desired. Likewise, antibiotics can be added to help maintain sterility. The pharmaceutical preparation according to the invention is divided into unitary dosage forms, for example in the form of ampoules containing 1 to 2000 mg of a pharmaceutically acceptable carrier per dosage form and about 1 to 20 mg, preferably about 3 to 15 mg, of the active ingredient (TPA, Pro-TPA or their mixtures) per dosage unit. Depending on the nature of the disease, as well as the age and condition of the patient, the daily dose to be administered to a patient of about 70 kg body weight will be in the range of 3 to 15 mg - preferably 5 to 10 mg - per 24 hours. The invention also relates to a process for the preparation of a pharmaceutical preparation which is characterized in that a biologically active protein according to the invention is admixed with a pharmaceutically acceptable excipient. The invention relates in particular to the processes for the preparation of the proteins described in the exemplary embodiments.

The following embodiments serve to illustrate the present invention without it being intended to be limited thereby.

Experimental part

Abbreviations used in the experimental part

BPTI basic pancreatic trypsin inhibitor

BSA bovine serum albumin

DEP diisopropylfluorophosphate

DMEM Dulbecco's Modified Eagle's Medium

DTT 1,4-dithiothreitol

EDTA ethylenediaminotetracetic acid

FCS fetal calf serum

PBS phosphate buffered saline 8 mM Na ₂ HPO ₄ ; 1.5 mM KH ₂ PO "; 0.14 M NaCl; 2.7 mM KCl)

RPMI-1640 Roswell Park Memorial Institute Culture Medium 1640

SDS sodium dodecyl sulfate

TCA trichloroacetic acid

Tris tris (hydroxymethyl) aminomethane

T-T (0.1) 0.1 M Tris · HClPH 9.1, containing 0.1% TritonX-100

Exemplary embodiment 1: application of the serum-independent cell line Bowes II

a) Creation of the Bowes II cell line using the conditioned medium collected from Bowes I cells A human melanoma cell line (Bowes RPMI 7272, described by DC Rijken and D. Collen, J. Biol. Chem. 256, 7035-7041 (1981) was obtained from Dr. E. Reich, Rockefeller University, New York The cell line is cultured in tissue culture flasks (150 cm ² , Coster) at 37 ° C CO ₂ in a humidified atmosphere of 95% air and 5% CO ₂ . The cells grow as adherent monolayers in Roswell Park Memorial Institute (RPMI) Medium-1640 (Gibco), which is replaced by sodium bicarbonate (2 g / l). Antibiotics (300 μg / ml

Penicillin; 200 μg / ml streptomycin; 10 ug / ml Tylozin) and 10% heat-inactivated (56 ⁰ C; 30 min fetal) calf serum (FCS) is added.

A tissue culture flask containing an adherent cell monolayer is selected. At confluency, the serum-containing medium is removed and replaced with 50 ml of the antibiotic-supplemented (see above) RPMI-1640 medium. No other additives are included in this medium. The cells are maintained at 37 ° C in humid atmospheric air which has been enriched with 5% CO ₂ . The medium is changed at 24-hour intervals. Initially the cells appear healthy and remain adherent. After 12 days, the cells begin to detach from the culture vessel and float freely in the medium. After 14 days, most of the cells have detached and only a small proportion of viable cells remain adherent to the flask. The medium containing the detached cells is removed. The sparse adherent culture of melanoma cells deprived of serum over a long period of time is covered with 50 ml of new medium composed of "conditioned medium" diluted with an equal volume of RPMI-1640. conditioned medium "is obtained as follows:

Se.rumhaltiges medium is taken from coherent serum-dependent melanoma cells Bowes-RPMI 7272 and replaced by RPMI-1640 medium alone. 24 h later, this medium is harvested, centrifuged for 5 min at 2000 U / min and enforced by a 0.45 μητι Milliporenfilter. The resulting solution is termed a "conditioned medium" and is immediately diluted with an equal volume of RPMI-1640

 Conditioned medium diluted with fresh RPMI-1640 is added to the culture at intervals of 4 to 5 days for a period of

3 months added. After this time, the cell count has increased considerably and the serum-free culture needs no further "conditioned medium." The culture is then fed solely with RPMI 1640 medium.

The serum-independent cells obtained in this manner are cultured in tissue culture flasks (150 cm ² , Costar) and must be seeded at a density of approximately 10 ⁶ cells / ml medium for survival purposes, typically the cells are seeded at 5 × 10 ⁷ cells / 50 ml RPMI-1640/150 cm ² pistons sown. The cells grow very slowly in the serum-free medium and show a generation time of about 6 days (compared to a generation time of about 24 hours in the serum-dependent cell line Bowes-RPMI 7272). In conflonence, the cells are detached by vigorous bursting of the flask to transfer adherent cells into the medium. The cells reacted in this mechanical manner are suspended at a concentration of about 10 ⁶ cells / ml in RPMI-1640 and used to re-order fresh tissue culture flasks. The cells not relieved by the regurgitation are supplied with fresh RPMI 1640 medium. In this way, a culture of the serum-independent cell line Bowes-Il is applied over a total period of 5 months. b) applying the Bowes H cell line in the absence of the conditioned medium collected from Bowes I cells

4 ml of a frozen stock of the serum-dependent melanoma cell line Bowes-RPMI 7272 (2.5 x 10 ⁶ cells / ml in DMEM) containing 15% fetal calf serum containing 10% DMSO are thawed and added to 15 ml of serum-free prewarmed DMEM in a 75-cm ² tissue culture flask added. The cells are incubated overnight at 37 ^° C. in humid atmospheric air supplemented with 5% CO ₂ . After this period, the entire medium, including non-adherent cells, is removed and replaced with 15 ml of serum-free DMEM. The incubation is continued until the cells appear granulated under the microscope (24 to 72 hours); At this point, 50% of the medium is removed and replaced with fresh serum-free DMEM. This procedure is repeated every two to three days as required (microscopic appearance of the cells, pH change of the medium), gradually increasing the total volume of the medium to 30 ml. After reaching 60 to 70% confluency (after about 3 weeks), the cells are transferred to two new equal size flasks, using 0.02% EDTA to detach the cells from the surface of the culture dish. At the first transfer, 40% serum-free DMEM is supplemented with medium taken from the culture prior to EDTA treatment. At this time, the serum-free line is established and the cells will continue to divide in the absence of conditioned medium, as long as a sufficiently high cell density, ie at least 30% confluence, is maintained.

Exemplary embodiment 2: Cultivation of Bowes II cells in submerged culture

Bowes II cells grown to confluence in tissue culture flasks and RPMI-1640 serum-free medium are detached by forceful bursting of the flasks by hand, combined to give 0.6 l of suspension at 2 × 10 ⁵ cells / ml and placed in a steam-sterilized 3 -l glass container transferred. The cell suspension is slowly agitated (40 rpm) with a mechanical stirrer. The temperature is adjusted to 37 ± 0.1 ⁰ C, and the culture is oxygenated by surface aeration with air containing 5% CO ₂ at a flow rate of 0.2 l / min. The drop in pH below 6.9 is prevented by resorting to clean air. During the initial adjustment period of about one week, fresh serum-free medium is added to prevent depletion of the glucose. The final culture volume is 1 liter. After reaching a cell density of 3 ... 4 x 10 ⁵ , the culture fluid is withdrawn at periodic intervals. Stirring is discontinued every 2 to 4 days for 3 hours to allow most of the viable cells to settle, whereupon the top 50% of the spent medium is withdrawn and replaced with fresh serum-free medium. Harvest fluids are centrifuged for 5 min at 2000 rpm (~ 300 g) to remove whole cells and cell debris. The solutions are stabilized with Triton X-100 · ® to a final concentration of 0.1% and acidified with acetic acid to a pH value of 5.5 ... 6.0 prior to storage at -20 ⁰ C.

Exemplary Embodiment 3 Cultivation of Bowes II Cells in Tissue Culture Flasks and Collection of Harvest Fluids

The serum-dependent cell line Bowes-II is incubated with 5 × 10 ⁷ cells / 150 cm ² tissue culture flasks (Costar) in 50 ml of RPMI-1640 supplemented with antibiotics (see Example 1) at 37 ^° C. in a humid atmosphere of 95%. Air and 5% CO ₂ inoculated. After adherence of the cells, the medium is collected from the cells and replaced with fresh antibiotic-supplemented RPMI-1640. The removal of serum-free harvest fluids is repeated at 24 hour intervals until the monolayer coalescence requires ablation. Confluence is reached after about 5 weeks. The removal takes place in the manner described in Example 1. The harvesting liquid is processed in the manner described in Example 2.

Exemplary embodiment 4: Determination of the content of TPA and Pro-TPA in harvesting liquids

Harvest fluids containing TPA and Pro-TPA are treated with ³ H-DFP (diisopropylfluorophosphate) of known specific activity. After incubation, the labeled enzymes are recovered and freed from unreacted radioactive DFP by making its precipitation and washing with trichloroacetic acid using the methods recommended in the literature for series proteases {JA Cohen et al., Method in Enzymology , Vol. Xl, p. 768 [1967]). This titration of the active sites of the activator causes the harvested harvested from serum-independent Bowes II cells to contain approximately 10 to 20 nmoles of total TPA per liter.

The release of total TPA (TPA and Pro-TPA) by Bowes I and Bowes II cells over 4 consecutive days can be determined as follows:

For 4 days, the harvest fluids are collected from Bowes I melanoma cells every 24 hours, which are grown in 75 cm ² tissue culture flasks (1.3 x 10 ⁵ cells / cm ² , 20 ml / flask) or are provided by Bowes -Il cells grown in 150 cm ² flasks (410 ⁵ cells / cm ² , 50 ml / flask). The activity of the plasminogen activator is measured in the ²⁵ I-fibrin assay (see below). In the case of Bowes II cells, TPA release remains constant throughout the four-day period, whereas in the case of Bowes i melanoma cells, TPA activity increases slightly during the first 3 days, then decreases on the fourth day. During this time, many of the cells had detached from the surface, whereupon RPMI supplemented with 10% FCS must be added to restore adhesion (see Table 1). In the ¹²⁵ l fibrin assay (EL Wilson and E. Dowdle, Int. J. Cancer 22, 390-399 [1978]), a solid phase substrate is created by exposing radioactive ²⁵ I-fibrinogen / fibrin as a thin layer the bottom inner surface of plastic tissue culture racks is deposited. Samples of the harvested fluids to be assayed are added to the racks, whereupon the TPA activity is measured as plasminogen-dependent solubilization of radioactivity with time. Since ¹²⁵ l fibrin determination measures both TPA and pro-TPA activity, the results on TPA activity given in Table 1 refer to the overall enzyme.

Table 1:

Total TPA release by serum-dependent Bowes I melanoma cells and serum-independent Bowes II cells in tissue culture flasks expressed in international urokinase (UK) units.

cell type Time (days) Total TPA (UK Unit) Bowes melanoma I 1 2 3 4 1 154 1 254 1 380 976 Bowes Il 1 2 3 4 3 560 3 660 3 950 3 850

In addition, the culture fluids of both the serum-dependent Bowes melanoma cells and the serum-independent Bowes II cells are examined for their individual content of TPA and Pro-TPA:

Bowes I melanoma cells are plated with 5 x 10 ⁵ cells per 35 mm replicate dish in 2 ml of RPMI containing 10% FCS. After 24 hours, the medium is replaced with serum-free RPMI and harvested every 24 hours for 3 consecutive days. At the end of each of the 24-h periods, the number of cells per plate is counted. Bowes II cells are plated at 3 x 10 ⁶ cells per 35 mm dish in 2 ml of RPMI.

Plasminogen activator activity is determined using the fluorometric assay using as substrate Cbz-Gly-Gly-Arg-AMC. The direct amidolylic effect of the TPA is measured fluorometrically by monitoring the fluorescence enhancement rate at 455 nm resulting from the amidolytic release of aminomethylcoumarin (AMC) from the fluorogenic substrate (see M. Zimmermann et al., Proc. Natl Acad., USA 75, 750-753 [1978]). 1 FU corresponds to the amount of enzyme that hydrolyzes 10 pmoles of substrate in one minute in the amidolytic assay. The results are shown in Table 2.

Table 2: TPA and Pro-TPA release by serum-dependent Bowes I. Melanoma cells and serum-independent Bowes II cells.

days Plasminogen activator release total TPA (%) Pro-TPA (%) ^b > in culture (FU / 10 ⁶ cells / 24 h) activity cell type cell density 1 (Cells χ 46.62 87.5 12.4 2 10 ⁵ xcm- ² ) 51.96 92.1 7.9 Bowes melanoma I 3 1.04 64.86 83.9 16.1 1 1.27 26,06 12.7 87.3 2 1.43 25.10 12.4 87.5 Bowes Il 3 3.88 24.40 , 10.9 88.9 3.88 4.01

a) The total TPA activity is measured after incubation of 295 μΙ harvest liquid with 5 μΙ plasmin (0.1 mg / ml) for 60 min at room temperature. For the determination, plasmin is inhibited by Trasylol®.

b) The amount of proactivator (Pro-TPA) is determined by subtracting the activity measured without piasmin activation from the total activity. '

As can be seen from Table 2, approximately 10% of the serum-dependent Bowes II cells secreted TPA are in the form of the active enzyme, while approximately 90% are in the form of the proenzyme (Pro-TPA). In contrast, the medium collected from serum-dependent Bowes I melanoma cells contains 90% TPA and approximately 10% Pro-TPA.

Exemplary embodiment 5: Preparation and purification of TPA and Pro-TPA

a) Preparation of a DE-3 Sepharose® column

26 mg of purified DE-3 inhibitor of Erythrina latissima (FJ Joubert et al., Hoppe-Seyler's Zeitschr Physiol. Chem. 302, 531-538 [1981]) are added to 5 ml of cyanogen bromide-activated Sepharose 4b® (Pharmacia) according to the instructions connected by the manufacturer. The matrix is equilibrated with O, 4M NaCl, 0.1% Triton X-100® and 0.02% sodium azide-containing phosphate buffered saline ("PBS") pH 7.4, and the matrix is then placed in a 5 ml tube. Pillar packed.

b) Chromatographic purification of emut fluids containing TPA and Pro-TPA on DE-3 Sepharose 4b® Two liters of harvested liquid obtained from serum-independent Bowes II cells (see Example 3) are adjusted to 0.4 M with respect to NaCl and to Triton-X -100® to a concentration of 0.1% and filtered through a 0.45 μηη membrane (Millipore). The harvest fluid is then the DE-3 ^Sepharose®: column (see above) was applied with a flow rate of 45 ml / h at room temperature and the effluent is discarded. After passing through the entire harvest liquid volume, the column is washed with about 50 ml of PBS containing 0.4 M NaCl and 0.1% Triton X-100®. The adsorbed proteins are then eluted using PBS containing 1.6 M KSCN, 0.4 M NaCl and 0.1% Triton X-100® followed by collecting 2 ml fractions at 4 ° C. The protein content of each of the fractions is determined by measuring the UV absorbance at 280 nm. It turns out that the adsorbed protein is eluted as a sharp peak. The fractions with the highest UV absorbance and highest fibrinolytic activity as determined in the ¹²⁵ l-fibrin are combined to give 8 ml of solution which is stored at -20 ^0C. This solution represents approximately 70-80% of the total activity applied to the column. The fractions of lower activity are summarized separately. The total activity in both summaries usually amounts to 90 ... 100%.

The stock is sampled. The protein is precipitated by adding trichloroacetic acid to a final concentration of 10% and subjected to SDS-polyacrylamide gel electrophoresis. The electrophoretogram shows a single protein band of approximate molecular weight 73,000 daltons as determined by the method of Weber and Osborne (J. Biol. Chem. 244,4406-4412 [1969]) using co-electrophoresed marker proteins known molecular weight.

The TPA and Pro-TPA content of the pooled stock is determined using the fluorometric determination with CBz-Gly-Gly-Arg-AMC as substrate (see Example 4). The total activity is measured after incubation with Plasming, which is inhibited for the determination with Trasylol®. The amount of Pro-TPA is calculated by subtracting the activity measured without piasmin activation from the total activity. It can be stated that the purification of the plasminogen activator from the medium of Bowes II cells yields a mixture of TPA and Pro-TPA in approximately equal proportions. Thus, during the isolation process, Pro-TPA is partially converted into the active enzyme, since in the unprocessed Emteflüssigkeiten collected from Bowes Il cells, the proportion of TPA 10% and the proportion of Pro-TPA is 90% (see Example 4, Table 2).

Treatment of emetic fluids from the serum-independent Bowes II cell line with low levels of whole calf serum (0.01%) results in conversion of Pro-TPA to TPA. This is most likely due to the presence of plasmin in the FCS.

c) Chromatographic purification of TPA- and Pro-TPA-containing Emte liquids on DE-3-Sepharose 4b in the presence of a proteinase inhibitor

Chromatography of Emte fluids derived from serum-independent Bowes II cells is performed in a manner similar to that described in Example 5b, except that basic pancreatic trypsin inhibitor (BTI) is included in the process at 0.1 KlU / ml becomes. Using the fluorometric determination with Cbz-Gly-Gly-Arg-AMC as substrate (see above), the TPA and Pro-TPA contents of the purified solution are determined; 90% of the TPA is in the proenzyme form and 10% in the active form. Thus, the conversion of Pro-TPA to TPA during the purification process is inhibited by BPTI.

-11 - zö ι aab

Embodiment 6: Demonstration of the presence of a Pro-TPA chain in purified TPA preparations

Harvesting fluids obtained from the serum-independent cell line Bowes II are purified as described in Example 5c by affinity chromatography on DE-3 Sepharose®. In the resulting purified solution, approximately 90% of the TPA is in the proenzyme form and approximately 10% is in the form of the active enzyme as evidenced by the amidolytic assay before and after the piasmine treatment. The solution is dialyzed in TT (0.1). Samples of this solution are mixed with equal volumes of PBS or 5 μg / ml plasmin-containing PBS. After incubation for 16 hours at 20 ° C SDS is added to a final concentration of 0.1%, after which the proteins are precipitated with 6% TCA. The precipitates of 200 ^ I samples of the original enzyme solution are washed in acetone and dissolved in 20 μ! 0.06 M Tris-HCl pH 6.8 containing 1% SDS and 10% glycerol was redissolved. Where necessary, these samples are reduced at this stage by adding 2 M DTT and μ11 30 min incubated at 37 ⁰ C. All samples are then boiled for 1 minute and 20 μΙ of each sample are electrophoresed in a 5 to 15% polyacrylamide gel 0.1% SDS plate. After electrophoresis, the gel is stained with Coomassie brilliant blue® and decolorized as usual. The electrophoretic lanes contain (a) molecular weight markers, (b) untreated and unreduced TPA solution, (c) untreated and reduced TPA solution, and (d) plasma treated and reduced TPA solution.

Lane (b) shows only one protein band with a molecular weight of approximately 73,000. Under reducing conditions (lane c), most of the TPA also migrates into the 73,000 dalton regime. However, additional weak bands can be observed in the 35,000 dalton region. Piasmin treatment and reduction with DTT convert the 73,000 dalton protein into two subunits with apparent molecular weights of 35,000 daltons and 38,000 daltons, respectively. These experiments demonstrate that the single chain Pro-TPA having a molecular weight of 73,000 daltons is converted by Piasmin treatment to the S-S linked two-chain form (TPA). TPA is then cleaved under reducing conditions to give the two subunits.

Embodiment 7: Determination of the enzymatic activity of Pro-TPA

Samples (500 μΙ) of a TPA preparation containing approximately 20 FU / ml total activator (TPA and Pro-TPA) in TT (0.1) are assayed in the presence (a and b) or in the absence (c and d) of 5 mM DFP Incubated at 2O ⁰ C for 60 min. Free DFP is removed by the method of HS Penefsky (J. Siol. Chem. 252, 2891-2899 [1977]) by centrifuging samples of 100 μΙ each of the reaction mixtures through 1 ml columns labeled TT (0, 1) contain exactly balanced Sephades G25®. These samples are volumes of 3 μΙ 0.5 mg / ml plasmin (b and d) or 3 μΙ PBS (a and c) is added, after which the samples are incubated for 30 min at 20 ⁰ C. Fifty microliters of each sample are then removed from the solution and added to 10 μΙ BPTI (1,000 KlU / ml) to inhibit piasm activity. Using the fluorometric assay, the amidolytic activity is determined in each of the samples. The results are shown in Table 3.

Table 3: Activation of DFP-resistant Pro-TPA with plasmin

Treatment activity

DFP plasmin

a) + 0.00

b) + + 8.23

c) -. - 6.25

d) - + 17.48

The sample has a total TPA content of 17.48 FU / ml (d), of which 6.25 FU / ml are in active enzyme form (c). Treatment with DFP (a) inhibits the effective enzyme to undetectable levels. After treatment with DFP, the active enzyme can be generated by incubation with plasmin (D), demonstrating that Pro-TPA is resistant to DFP treatment and lacks moderate enzyme activity.

Embodiment 8 Separation of Pro-TPA from TPA

The solution of TPA (10%) and Pro-TPA (90%) obtained according to the description in Example 5c is adjusted to pH 8.0 with 0.1% Triton X-100® containing 0.1 M Tris. HCl and measured with respect to DFP adjusted to 1 mM. After four hours of incubation at 37 ° C., the mixture is passed through a 5 ml DE-3 Sepharose® column (see Example 5a). The effluent containing the irreversibly inhibited TPA is discarded. The column is washed with 6 column volumes of PBS containing 0.4 M NaCl and 0.1% Triton X-100®, then eluted as described in Example 5b with PBS containing 1.6 M KSCN, 0.4 M NaCl and 0.1% Triton X-100®. The fractions with the highest UV absorbance are summarized. The resulting stock contains Pro-TPA in substantially pure form as no amidolytic activity is detectable in the fluorometric assay using Cbz-Gly-Gly-Arg-AMC as the substrate. Both amidolytic and fibrinolytic activity can be restored by treatment with plasmin, which converts pro-TPA into the active enzyme.

Embodiment 9: The binding of TPA and Pro-TPA to insolubilized fibrin

Five samples containing TPA and Pro-TPA in different proportions are recovered as described in Example 5c or by converting Pro-TPA to TPA in such a solution. Their contents of active TPA and Pro-TPA are determined from the results of the fluorometric determination before and after the piasmin treatment described in Embodiment 4. These samples are then diluted in TT (0.1) so that, in the presence of 2 μg plasminogen, 0.2 ml will contain approximately 30-50% of the ¹²⁵ l fibrin applied to the bottom of linbro racks during the course

of 1 hour.

Aliquots (0.2 ml) of the samples are added in quadruplicate to linbro racks coated with ¹²⁵ l fibrin (30 mg, 100,000 cpm). After incubation at ⁰ ° C for ¹ hour, two racks of each quadruple set are washed three times with TT (0.1) to remove unbound TPA proteins.

The binding activity is measured to be the amount of ¹²⁵ l-fibrin which has been solubilized in 1 h after adding Trie.HCl pH8.1 containing 0.3 ml of 2 μg plasminogen and 80 μg BSA. The total TPA activity added to the racks is measured as the amount of ¹²⁵ l-fibrin that did not wash within one hour after adding 2 μg plasminogen and 80 μg BSA (0.3 ml of the same buffer) to racks been solubilized. The results are shown in Table 4.

Table 4: The binding of Pro-TPA to insolubilized fibrin

sample

, Pro-TPA

Fibrinolytic activity {% I-fibrin solubilized in 60 min)

total activity

Activity after the wash

ι bound

88.7 34.0 64.0 87.0 88.0

29.83 45.90 56.56 48.88 53.85

29.48 98.8 37.52 81.7 41.53 73.4 48.76 99.8 52.49 97.5

Table 4 shows that a greater percentage of total TPA enzyme is bound to insolubilized fibrin when the TPA preparation is in the PrO activator form.

Embodiment 10: Pharmaceutical preparation for parenteral administration

A solution containing TPA and / or Pro-TPA obtained as described in Example 5b, 5c or 8 is dialyzed against 0.3 molar sodium chloride containing 0.01% Tween 80® and stored at -80 ° C. Prior to administration, the concentration is adjusted to 75 μg / ml total TPA (i.e., TPA or Pro-TPA or TPA plus Pro-TPA) and 0.3 M NaCl. The solution is sterilized by filtration through a 0.22 μm membrane filter.

This procedure is suitable for the preparation of solutions of TPA, Pro-TPA or TPA plus Pro-TPA for both parenteral and intravenous administration.

Exemplary embodiment 11: Selection of a mutant serum-independent cell line Bowes II which is capable of producing increased TPA levels

5% fetal calf serum is added to a Petri dish culture of the Bowes-lI serum-independent cell line in order to increase the growth rate of the cells and to ensure that more cells are in the s-phase at a certain time. At the time of rapid cell division as well as in the exponential growth phase and when approximately 70% confluency is reached, 0.1 mM N-methyl-N-nitro-N'-nitrosoguanidine (MNNG) is added to the culture. This concentration causes the death of approximately 70 to 80% of the cells. After 36 hours, the remaining viable cells are removed from the Petri dish by trypsinization. The cells are reseeded at a density of 1 × 10 ⁵ cells / 60 mm Petri dish. Two weeks later, colonies of cells are observed on the bottom of the dish, each colony representing the progeny of a simple cell surviving the genesis. These colonies are then covered with 1 ml of an overlying solution composed of:

50 μΙ plasminogen (1 mg / ml),

300 μΙ 8% casein in isotonic saline,

600 μΙ 2.5% agar in isotonic saline solution as well

800 μM RPMI-1640 medium.

Around the TPA-producing clones, lysis areas develop, with the largest lysine areas observed around those clones producing the most TPA. The highest TPA-producing clone is isolated and expanded on a Petri dish.

Claims (14)

Invention claim: 1. A process for the preparation of TPA, Pro-TPA or mixtures thereof and optionally the usual pharmaceutical application forms, characterized in that for the production of TPA and / or Pro-TPA capable serum-independent human cells cultured in a serum-free medium and the desired proteins from the harvesting liquid is isolated and characterized further characterized in that - if desired - a recovered mixture of TPA and Pro-TPA is separated or converted into the individual components and optionally TPA, Pro-TPA or mixtures thereof with conventional galenic auxiliaries and / or carriers to usual be processed pharmaceutical applications. 2. Method according to item 1, characterized in that the serum-dependent human cells are Bowes II cells or their mutants. 3. The method of item 1, for the preparation of Pro-TPA-free TPA, characterized in that the present in a mixture obtained Pro-TPA is enzymatically converted to TPA. 4. The method according to item 1 for the preparation of TPA-free pro-TPA, characterized in that during the isolation and purification procedure a protease inhibitor is included and that the final purification is carried out in the presence of an inhibitor which selectively inhibits TPA. 5. The method according to item 1, characterized in that it is a process for the preparation of mixtures of tissue plasminogen activator. 6. The method according to item 5, characterized in that it is a process for preparing a mixture of tissue plasminogen activators, wherein 90% in the proenzyme form and 10% in the form of the active enzyme. 7. The method according to item 3, characterized in that a recovered mixture of TPA and Pro-TPA is treated with plasmin or an enzyme which exerts an equivalent effect and pro-TPA. 8. The method according to item 4, characterized in that the protase inhibitor is selected from aprotinin and basic pancreatic trypsin inhibitor. A process according to any one of items 4 or 8, characterized in that the final purification is carried out by chromatography on a column containing a selective affinity reagent in the presence of diisopropylfluorophosphate or nitrophenylguanidinobenzoate. 10. The method according to item 1, characterized in that the final purification is carried out by chromatography on a monoclonal anti-TPA antibody column. 11. The method according to item 1, characterized in that the cells are cultured as an adherent culture. 12. The method according to item 1, characterized in that the cells are cultured as a suspension culture. 13. The method according to item 1, characterized in that the harvested harvesting liquid is centrifuged and then subjected to affinity chromatography. 14. The method according to item 1, characterized in that a Datergen is included during the isolation and cleaning process.