AU6336986A - Methods for the recovery of tissue plasminogen activator - Google Patents

Description

METHODS FOR THE RECOVERY OF TISSUE PLASMINOGEN ACTIVATOR FIELD OF THE INVENTION

This application is a continuation-in-part of United States Patent Application Serial Number 773,334, filed on September 6, 1985, now abandoned.

This invention relates to the recovery of tissue plasminogen activator (t-PA) from liquid media and more specifically, to an improved method for recovering intact single-chain t-PA substan¬ tially free of degraded t-PA and other non-homologous proteins.

BACKGROUND AND PRIOR ART

Plasminogen activators have received attention for their role in the fibrinolytic system. These enzymes catalyze the conversion of the proenzyme plasminogen into the proteolytiσ enzyme plasmin; plasmin can, in turn, degrade fibrin, a major component of blood clots. Thus, plasminogen activators are potentially useful for the therapeutic treatment of blood clots.

The known plasminogen activators include streptokinase, which is of bacterial origin, urokinase (u-PA) , which has been isolated from urine and culture fluids, and tissue plasminogen activator (t-PA) , which is now becoming available from cultured human cells (Rifkin et al. , J. Exp. Med. 139:1317-1328 (1974); Wilson et al. Cancer Res. 4_0:933-938 (1980)). Streptokinase and u-PA are available commercially, but appear not to possess the therapeutic efficacy of t-PA. Intact t-PA is a glycoprotein having a molecular weight of about 66,000 daltons, and exists as either a one chain polypeptide (Binder et al., J. Biol. Chem. 254:1998-2003 (1979)) or it may be cleaved by plasmin (Wallen et al. , Prog, in Fibrinolysis 5_:16-23 (1981)), into a two-chain form, wherein the two polypeptides are linked by a disulfide bond (Rijken et al. , Biochem. Biophys. Acta 580:140-153 (1979)). Non-glycosylated, enzymatically active t-PA has been produced in eukaryotiσ cells grown- in the presence of drugs that prevent glycosylation (Little et al. , Biochemistry 23:6991-6995 (1985)); and in bacteria (Pennica et al. , Nature (London) 301:214-221 (1983)). Degraded forms of t-PA, having molecular weights of approximately 50,000 and 32,000, have been found coexisting with intact, one- chain and two-chain t-PA (Granelli - Piperino & Reich, J. Exp. Med. 148:223-234 (1978)). Prior art methods for isolating t-PA have not been particularly effective at separating the degraded forms of t-PA from the intact t-PA.

In pharmaceutical formulations of t-PA, the availability of substantial quantities of pure intact single-chain enzyme is important and desired. The strong fibrin binding exhibited by tr-PA (Thorsen et al. , Throm. Diath. Haemorrh. 2_8:65-74 (1972)) is believed to be important for its therapeutic efficacy. The lower molecular weight degraded forms, which have aberrant fibrin binding properties (Banyai et al. , FEBS Lett. 163:37-41 (1983)), do not appear to display the specificity and clot localization properties of intact one-chain and two-chain t-PA. Further, it is believed that single-chain t-PA is more desirable in pharmaceutical formu¬ lation than the two-chain form due to the much slower rate at which the single-chain form is inactivated by specific inhibitors of t-PA found in plasma (Lecander et al., Brit. J. Haematol. 5χ:407-412 (1984)).

Various protocols have been described for the purification of t-PA using chromatographic, electrophoretiσ, and selective extraction and precipitation methods. Most of these methods, including a widely used purification (Rijken and Collen, J. Biol Chem. 256:7035-7041 (1981)), are not appropriate for the large- scale production of t-PA as they are inefficient in product recovery, only partially effective in removing impurities, or use adsorbants which may introduce toxic, mitogenic, tumorogenic or immunogenic ligands into the t-PA preparation (Reagan et al. , Throm. Research 40:1-9 (1985)). Large scale purification methods employing immunoaffinity chromatography (Wallen et al Eur. J. Biochem 131:681-686 (1983); Nielsen et al, EMBO J. 2_:115-119 (183)) are limited by the cost of the antibody resin, the difficulty in sterilizing or sanitizing this resin and by the potential for the antibody or fragments of the antibody leaching into the recovered t-PA. In addition, the published methods do not provide procedures to concentrate t-PA to give useful therapeutic formulations. Furthermore, the presence of degraded forms of t-PA in preparations of the purified enzyme remains problematic to those skilled in the art (Kruithof et al. , Biochem. J. 226:631-636 (1985)). Degraded t-PA is commonly found in fermentation broth. Degraded t-PA not only dilutes the intact t-PA, but in addition, as mentioned above, is not specific and is less: able to localize clots as the intact t-PA. Therefore, contamination of final t-PA product with degraded t-PA provides serious drawbacks to the product as a therapeutic agent. However, chromatographic methods for the specific recovery of intact t-PA free from degraded forms have not been known, so that the method disclosed by Rijken and Collen, supra, fails to separate intact t-PA from its degraded forms, and the two forms have, consistently co-purified together.

Most tissue culture cells require serum supplementation of media for optimal growth and survival. The known methods for recovery of t-PA from conditioned tissue culture media are generally effective only when serum-free media is used. In those examples wherein serum containing production medium is used (Reagen et al, supra; Cederholm-Williams & Porter, Brit J. Dermatology 110:423-429 (1984), Kluft et al. , Adv. Biotechnol. Processes 2:97-110 (1983)) only partially pure t-PA or t-PA containing degradation products is recovered. This degradation is attributed to serum components and may be only partially blocked by the addition of proteinase inhibitors (Reagen et al, supra) . SUMMARY OF THE INVENTION

The present invention provides a rapid, efficient method for the recovery of intact, single-chain tissue plasminogen activators (t-PA) from liquid media, e.g., serum-free and serum-supplemented media used to culture cells which secrete intact t-PA or from extracts of cells which intracellularly deposit t-PA or non- glycosylated t-PA polypeptide. The novel method of the present invention effects the recovery of t-PA substantially free of degraded t-PA by contacting a liquid medium with at least one substrate capable of effecting a separation of intact t-PA from degraded t-PA.

The present invention also provides methods for further adsorbing t-PA onto additional adsorbant substrates, e.g. adsorbant substrates comprising at least one aminocarboxylic acid, followed by eluting and recovering the t-PA. Such additional adsorption and elution can precede or follow the novel methods, while retaining the benefits of the present invention.

The present invention also provides a method for minimizing the amount of degraded t-PA and two-chain t-PA recovered from serum- or serum fraction-supplemented media by pre-treating the serum with an additional substrate such as, e.g., lysine-Sepha- rose (Pharmacia Fine Chemicals, Pisσataway, N.J.) chromatography.

Untreated serum used in growth media for culture cells contains plasminogen and plasmin which are known to proteolytically cleave t-PA (Wallen et al, supra) . Lysine- Sepharose chromatography has been shown to be effective in the removal of these proteins from serum (Wu et al, Exp. Cell Research 9_6_:37-46 (1975) Quigley et al J. Biol. Chem. Vol. 249, pg. 4306-4311 (1974)). Such depleted serum is capable of supporting the growth of tissue culture cells (Wu et al, supra; Kaufman et al, Molec. Cellular Biology 5:1750-1759 (1985)). The present invention provides improved methods for the removal of plasminogen and plasmin from serum, and further provides a novel use of "scrubbed serum" in combination with aprotonin (an inhibitor of t-PA proteases) as an essential reagent if intact single-chain t-PA is to be recovered from serum supplemented media.

Also provided are compounds and compositions obtained by practicing the present invention, said compounds and compositions comprising intact t-PA, substantially free from degraded t-PA and other unrelated proteins, as well as methods for using such compounds and compositions.

One substrate useful in the present invention, Zn chelate, has previously been employed for recovering t-PA (Rij en et al. , supra.) However, the prior art protocols diff r significantly from those disclosed here. The modified zinc column protocol disclosed here provides the advantages of better separation of intact from degraded t-PA, and increasing the efficiency of purification by separating the bulk of the contaminating proteins, as well as the degraded t-PA, from the desired single-chain t-PA.

The literature teaches the use of high ionic strength solu¬ tions for chromatography, greater than 0.5 M salt concentrations when using metal-chelate resins to minimize non-specific adsorptio effects. (Rijken et al, supra; Porath et al. Nature 258:598-599 (1975)). The present invention includes the unexpected observatio that the use of a low ionic strength washing condition (under 100 mM salt, and preferably NaCl) allows for the elution of degraded t-PA and the majority of other proteins bound to the column while retaining intact t-PA. This results in the ultimate recovery of t-PA free of degraded t-PA and unrelated proteins which is not possible if traditional methods (Rijken et al, supra, Rijken & Collen, supra) are used.

An additional substrate useful in certain embodiments of the present invention, immobilized lysine, has also been used to recover plasminogen activator activity from human plasma and homogenized human veneous tissue (Radcliffe and Heinze, Arch. Bioche . Biophys. 139:185-194 (1978)), cadaveric perfusates (Allen and Pepper, Thrombos. Haemostas. 4_5:43-50 (1981), and from medium conditioned by incubation with a guinea pig tumor cell lin (Oerstein et al. , Cancer Res. 4_3_:1783-1789) ) . This substrate, however, has been reported ineffective for the purification of t- found in human uterine tissue (Rijken et al. , supra) . Previously, the identities of the isolated activators were not rigorously determined, nor were the purities of the enzymes established. Further, the previously reported methods for elution of t-PA from the immobilized lysine substrates did not provide a system to concentrate t-PA. It is important to obtain t-PA in concentrations useful for therapeutic formulation and subsequent administration. The present invention provides a method for recovering t-PA from lysine-Sepharose in very pure form, using either basic or acidic eluting conditions. Acidic elution provides a product with higher solubility which is more suitable for pharmaceutical formulation. This formulation provides methods for concentrating the t-PA which include alone, or in combination, dialysis, diafiltration, cationic exchange chromatography on S-Sepharose, and freeze-drying.

One possible detergent used in purification, Zwittergent 3- 12 (Calbiochem, La Jolla, California) , can be removed from t-PA by dialysis or diafiltration. Alternatively, Pluroniσ ^m F-68 (BASF) can be used. Either of these detergents have the desireable property that they can be freeze-dried to a powder along with the t-PA.

It is thus an object of the present invention to provide a rapid, simple method for the recovery of tissue plasminogen activators which increases the recovery of intact t-PA, substan¬ tially free of degraded t-PA and other undesirable proteins and polypeptides, from a variety of liquid media such as those used in the culture of eukaryotic or bacterial cells, or from extracts of such cells, which express the intact t-PA polypeptide.

It is a further object of the present invention to provide a method which maximizes the amount of single-chain enzyme relative to the amount of two-chain recovered.

It is a further object of the present invention to provide a method for the recovery of intact t-PA substantially free of other proteins including other plasminogen activators, such as u- PA, and non-homologous proteins.

It is a yet another object of the present invention to provide a method for the recovery of intact t-PA which provides a product suitable for the subsequent formulation as an effective pharmaceutical composition for therapeutic use.

It is yet another object of the present invention to provide a method for formulating t-PA in a concentration suitable for therapeutic use.

It is yet another object of the present invention to provide a method for formulating t-PA in a concentration suitable for therapeutic use.

It is yet another object of the present invention to provide a method for formulating t-PA useful for large scale commercial production of the desired form of t-PA. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows chromatography studies of tissue plasminogen activator. Conditioned serum-free medium or medium supplemented with serum which had been pretreated by adsorption with lysine- Sepharose was clarified and applied to a column of Zn-chelate *τrt

Sepharose . This column was developed as described in the text of Example 1. Figure (A) shows the elution pattern of total protein (A 280 nm) and t-PA activity (histograph) . A 5-50 micro- liter (ul) aliquiot of each fraction was incubated at 37° C with 200 ul of 0.01 M Tris-HCl (pH 8.5), 0.1% Tween 80 and 0.2 mM S-

2288J (Kabi) . The change in adsorbancy at 405 nm was monitored to measure the amidolytic activity of t-PA. The t-PA contained

+"τn in the "Zn B" fractions was applied to a lysine-Sephaose column, and eluted either at pH 8 (Figure B) or at pH 4.0 (Figure C) as described in the text of Example 1. In each of the figure panels the arrows at the top indicated the application of a different wash or elution buffers to the columns.

Figure 2 shows an SDS - polyacrylamide gel electrophoresis of tissue plasminogen activator. The figure shows a coomassie blue stained gel (Laemmli, Nature (London) 227:680-685 (1970)) of three independent preparations of t-PA recovered using the procedures described in Example 1 from conditioned medium supplemented with pretreated serum. The left most lane contains a mixture of reduc and alkylated standard proteins, from top to bottom: phosphorylase b (94,000 mw) , albumin (67,000 mw) , ovalbumin (43,000 mw) , carbonic anhydrase (30,000 mw) . The remaining lanes each contain 5mg of tissue plasminogen activator. Lanes marked with a (+) contain t-PA which had been chemically reduced with DTT before electrophoresis.

Figure 3 shows a zymograph of t-PA recovered by a method of the present invention from Zn-chelate Sepharose. Each lane contains one unit of t-PA. The samples were mixed with Laemmli sample buffer (no DTT) , but not heat denatured, and electrophoresed at 4°C through a 0.75 mm thick 8.7% SDS -γn polyacrylamide gel using the Hoeffer "Mighty Small " electrophoresis unit. Electrophoresis was carried out at a constant 150 V. After electrophoresis, the gel was soaked for 15 minutes each in two changes of 100 ml of phosphate buffered saline (PBS) + 2.5% (v/v) Triton X-100, followed by two washed with PBS. The gel is placed onto a standard plasminogen-enriched fibrin plate and incubated at 37°. Zones of clearing are detected within 2 hours. Lane "A" was obtained from samples eluted with 20 mM Tris-HCl (pH 7.5), 25 mM NaCl, 0.1 M imidazole, 0.01% Tween 80 (termed Zn A), and indicates more rapidly migrating (i.e., degraded) t-PA near 50,000 and 32,000 daltons. Lane "B" was recovered by elution with 20 mM Tris-HCl (pH 7.5), 1.0 M NaCl, 50 mM NaEDTA, 0.01% Tween 80 (termed Zn B) .

Figure 4 shows the separation of intact and degraded t-PA. The figure shows a commassie blue stained gel of a non-reduced sample of partially purified t-PA which contained intact (65,000 mw) and degraded (50,000 and 32,000 mw) t-PA ("Load") and samples in which a substantial separation of these forms into the "A pool" ("Zn A") and "B pool" ("Zn B") had been effected through chromatography on Zn-chelate Sepharose using the protocols described herein. Other experimental details were as described in:Figure 2.

Figure 5 shows the inhibition by aprotinin of the conversion of:one-chain to two-chain t-PA in various tissue culture media. Increasing amounts of aprotinin were added to tissue culture media used for the production of t-PA. The t-PA synthesized during 48 hours of incubation who analyzed by "Western Blot" analysis as described in the text. A shows t-PA produced in serum-free medium; B, medium supplemented with 0.5% serum, and C, medium supplemented with 0.5% serum which had been preadsorbed with lysine Sepharose.

DETAILED DESCRIPTION

A rapid, efficient procedure has been developed for the recovery of intact, single-chain tissue plasminogen activator (t-PA) from a liquid medium. The method of the present invention comprises contacting liquid medium which contains t-PA with at least one substrate capable of effecting a separation of intact t-PA from degraded t-PA, and with additional substrates capable of effecting a separation of the intact t-PA from other unrelated proteins.

The present invention also provides methods for treating serum, which is to supplement the nutrient medium used for the production of t-PA by tissue culture cells, by contacting this serum with lysine-Sepharose. This pre-treatment was found to be essential to minimize the proteolytic degradation of t-PA and further effects the removal of serum proteins which otherwise co- purify with t-PA.

The present invention also provides compounds and compositions obtained by practicing the present invention, as well as compounds and compositions comprising intact t-PA, and other unrelated proteins and methods for their use.

The liquid media used in one aspect of the invention have generally been conditioned by incubation with cells which actively produce intact t-PA, herein exemplified by, but not limited to, a Bowes melanoma cell-line which has been genetically engineered to express higher levels of t-PA than does the parental cell line. Any eukaryotic or procaryotic cell culture or cell line which secretes t-PA or non-glycosylated t-PA, such as tunicamycin treated Bowes melanoma cells (Little et al. , supra) , or lysates of cells, such as E. coli (Pennica et al, supra) , which deposit the t-PA or the non-glycosylated t-PA polypeptide intracellularly, would be appropriate conditioning agents for liquid media useful in the present invention. Such liquid media will generally contain a mixture of intact frPA and degraded t-PA. Degraded t-PA includes those forms of t- PA which have been proteolytically cleaved to produce lower molecular weight forms, such as the 50,000 and 32,000 species. Also included are those forms of t-PA which have been modified to alter their fibrin binding or fibrin activation characteristics, resulting in decreased thro bolytic activity or decreased specificity.

Ligands employed in the present invention are capable of effecting a separation of intact t-PA from degraded t-PA.

Examples of such ligands include an adsorbant substrate comprising the general formula:

support- (CH.) - substrate where n is greater than or equal to zero. These molecules chelate metal ions such as Zn , Cu , Ni or Co . Other chelating agents capable of complexing divalent cations may be useful in the present invention as well.

Additional benefits can be obtained in the practice of this invention by employing a plurality of ligands, such as lysine and propylsulfonate to further separate intact t-PA from undesirable contaminants.

For ease of use, the ligands effecting separations are generally immobilized on support substrates. These support substrates can comprise any support materials known to the art which do not interfere with the separations as disclosed herein. Such support substrates can be linked, e.g., convalently bound, to the separation ligands by any conventional means to provide increased ease in handling and washing such substrate to improve the efficiency of the method of the present invention. Support substrates known to the art include dextrans, agarose, cellulose, polyacrylamide, silica, etc. When an adsorbant substrate is linked' to a support substrate, the term resin is used.

Certain preferred embodiments of the present invention produce higher yields of intact one-chain t-PA, substantially free from intact, two-chain t-PA and degraded t-PA. In the preferred embodiment, the liquid medium is serum-free nutrient medium incubated with Bowes melanoma cells. This medium usually contains low levels of degraded t-PA and unrelated proteins in mixture with intact t-PA. However, tissue culture cells frequently require for optimal growth or viability media with serum, fractionated serum, or defined proteins, such as albumin, transferrin, insulin, cell attachment, growth factors, etc. It is reported in the literature (Reagan et al, supra; Cederholms- Willia s and Porter, supra; Kluft et al, supra) and observed by us that the presence of serum in the medium used for the production of t-PA results in increased levels of degraded and two-chain t-PA or decreases the purity of the t-PA recovered. In the preferred embodiment of the present invention where serum or fractionated serum was used to formulate the liquid medium, it was generally pretreated by adsorption to lysine- Sepharose. This pre-adsorbed serum supported survival and growth of the cell cultures equivalent to untreated serum (Wu et al, supra; Kaufman et al, supra.) This pre-treatment removed sub¬ stantially all the plasminogen or plasmin from the serum (Deutsc and Mertz, Science 170:1095-1096 (1970). Plasminogen, when converted to plasmin by plasminogen activators, is known to catalyze the degradation of t-PA (Banyai et al, supra; Wallen et al. , Prog. Chem. Fibrinolysis Thrombolysis 5 :16-23 (1983)). This removal of plasminogen was essential for the recovery of high yields of single-chain t-PA. It is reported that the inclusion of protease inhibitors in t-PA production medium is only partially effective in preventing the degradation of t-PA (Reagan et al, supra) . We have furthermore observed tha the pre-treatment of serum removes other materials having affinity for lysine, and which may otherwise co-purify with the t-PA in certain embodiments of the present invention. The use o pre-adsorbed serum is therefore essential for the recovery of intact t-PA free of degraded t-PA and other unrelated protein from serum-supplemented medium.

As an example, the pre-treatment of serum was accomplished by first diluting the serum with three volumes of cold sterile water. The diluted serum was passed at 4°C through a column of lysine-Sepharose resin at a flow rate of about one column volume per hour. The effluent, herein referred to as "scrubbed serum", was collected, assayed for plasminogen (Wu et al, supra) , filter sterilized and stored frozen until used in the formulation of th liquid medium. Approximately one milliliter of resin was used t treat each milliliter equivalent of undiluted serum. The level of plasminogen in sera varies significantly. It therefore is sometimes necessary to use amounts of resin greater than that specified above. With all serum tested, it was found that less resin was required for the complete removal of plasminogen if th serum is diluted as described here, than if undiluted serum is used as described in the literature (Wu et al, supra) . It may b necessary with the diluted serum to adjust the osmotic strength by adding NaCl before using it in to supplement tissue culture media. The resin is then regenerated by washing it with a solution comprising 5 M urea, 1 M NaCl, 50 mM Na EDTA (pH 7.5), followed by sterile water. The resin column was sanitized by washing with 20% ethanol and then storing the column with ethano for at least 18 hours. The resin was thoroughly washed with sterile water before re-use.

To further exemplify a presently preferred embodiment of on aspect of the present invention, Bowes melanoma cells, adsorbed to tissue culture flasks (Rijken and Collen, supra) or microcarriers (Kluft et al. , supra) were used to condition liqui media which contained 0 to 0.5% scrubbed serum. Aprotinin at a concentration of 5 to 100 KlU/ml, and typically 10 KlU/ml was included in the t-PA production medium. The cells were removed by centrifugation or filtration. Filters used for clarification should be of low-protein binding materials. It is useful to pre-

^■"ττι treat the filters by passing a solution of 0.1% Pluroniσ F-68 (BASF) or Tween 80 (Atlas Chemical Company, Inc.) therethrough. These conditioned media were chilled to approximately 4°C, adjusted to between approximately pH 7 and 8 with 1 M HC1 or NaOH, supplemented with 0.01% (w/v) Tween 80 or Pluronic F-68 and passed through a first column comprising Zn Chelate Sepharose ^m or Zn Chelate Fast Flow ^m resin. These resins were prepared as recommended by the manufacturer.

Routinely, the t-PA from 200 liter of 0.5% serum- supplemented conditioned medium could be completely adsorbed onto 1 liter of resin. Medium may be passed over the resin at the maximal flow rate recommended by the manufacturer, with substantially all the detectable t-PA activity retained on the resin.

The column employed in this embodiment of the present invention desirably has a high binding capacity and flow properties such that the t-PA could be rapidly concentrated from the culture medium. Desirably, the medium should be passed through the column without significant depletion of essential nutrients, modifications of pH or ionic strength nor addition of compounds toxic to tissue culture cells, so that the medium may be recycled into the culture, thus reducing the production costs related to media use. Optimal binding and recovery of t-PA was achieved when chromatography was performed at 4°C using buffers of approximately pH 7-8, e.g., 20 mM Tris-HCl (pH 7.5 measured at 20°C) , and supplemented with 10 KIU aprotinin/ml and with 0.01% (w/v) Tween 80 or Pluronic F-68.

The t-PA-charged resin was washed with buffer containing approximately 1.0 M NaCl to remove non-specifically adsorbed material, and then with a buffer containing approximately 25 mM NaCl to decrease the ionic strength of the aqueous phase of the resin. The decreased ionic strength of the aqueous phase of the intermediate washes, generally less than the equivalent of 100 m NaCl, and desirably below 25 mM NaCl, is an important feature of the embodiments of the present invention employing metal chelate adsorbant substrates such as Zn ++ chelate. It has been discovered that, employing medium at the ionic strengths taught by the prior art (1 M NaCl) (Rijken et al, supra;

Rijken & Collen, supra) , intact t-PA is not separated from degraded t-PA or from the bulk of protein adsorbed to the column

At the ionic strength of 25 mM NaCl, degraded forms of t-PA and the bulk of the non-related proteins adsorbed to the resin can b eluted as described below without significant elution of the desired intact form of t-PA. Plasminogen activators which have been adsorbed during the practice of the present invention can be eluted from the substrate. When employing an adsorbant substrate, an agent which is capable of disrupting the adsorption will be useful. It is considered desirable to elute t-PA or other proteins by means of an agent which competes for the binding sites on the adsorbant. For example, t-PA adsorbed to an adsorbant substrate comprising a metal chelate such as zinc chelate can be eluted with imidazole, hiatidine or zinc, among others. Elution can also be effected by such means as salt concentration, pH, or the use of chelating agents such as sodium ethylenediaminetetraacetic acid (NaEDTA) . The. selection of the eluting agent and precise conditions, i.e., pH, ionic strength, temperature, are chosen so that the selectiv elution of degrated and intact t-PA are achieved thereby.

Plasminogen activator activities characterized by molecular weights of approximately 50,000 and 32,000, as determined by zymαgraphy (Granelli-Piperino & Reich, supra) , using plas inogen containing fibrin indicator plates, were eluted by washing the resin with 25 mM NaCl which contained an agent capable of disrupting the adsorption of these species, e.g., 100 mM imidazole. These plasminogen activators could be specifically inhibited and immunopreσipitated by monoclonal antibodies directed against t-PA and therefore appear to be degraded t-PA. u-PA is also eluted from the resin by this procedure. Since man tissue culture cells secrete u-PA, this chromatography procedure ensures the recovery of t-PA free from this plaminogen activator which possesses less fibrin-clot specificity.

The decreased ionic strength of the aqueous phase of the intermediate washes, generally less than the equivalent of 100 m NaCl, and desirably below 25 mM NaCl, is an important feature of embodiments of the present invention employing metal chelate adsorbant substrates such as Zn ++-chelate. The prior art teache the use of high ionic strength solutions to minimize non-specifi ionic interactions of proteins with metal chelating resins. We have suprisingly found that the resolution of this resin is greatly enhanced by use low ionic strength solutions. We have found that using low ionic strength solutions of less than 100 m

NaCl or similar salts, degraded t-PA and the bulk of unrelated proteins adsorbed to the Zn -chelate resin can be eluted while retaining most of the intact t-PA adsorbed to the resin. This allows for the final recovery of t-PA essentially free of degraded t-PA and for the production of t-PA of greater purity than is possible had the method for chromatography of t-PA on

++ Zn -chelate resin in the prior art been used (Ripken et al, supra; Rijken & Collen, supra) .

Complete elution of the adsorbed intact t-PA free of the degraded t-PA was effected by washing the resin with 1 M NaCl, 5 mM Na EDTA. Alternatively, the eluting buffer can contain 1.0 M

NaCl, 100 mM imidazole or gradually increasing amounts of NaCl (0.025 to 1.0 M NaCl) with 100 mM imidazole. The latter results in the successive elution of t-PA subpopulations, distinguished by their differing affinities for the resin under the conditions of increasing ionic strength. In a preferred embodiment, NaEDTA effects the highest recovery of t-PA from the adsorbant substrate.

The intact t-PA recovered from the Zn -chelate resin can be further treated to remove additional, unrelated contaminants. For., example, the intact t-PA recovered from the Zn -chelate resin was then passed though a second column comprising an aminocarboxylic acid (e.g., lysine) linked directly or via a spacer (e.g. a six carbon aliphatic spacer) to a support substrate (e.g. Sepharose) . However, certain benefits of the present invention can be obtained with any compound wherein both an amino and σarboxyl group are free to interact with t-PA.

Such compounds include, e.g., 3-amino-n-proprionic acid, 4- amino-n-butyric acid, 5-amino-n-heptanoic acid, 6-amino-n- hexanσic acid, among others. Included also are cyclic compounds such as tranexamic acid, and other analogs of lysine, such as aminoethylcysteine, lysopine and octopine, which may possess affinity for t-PA. Such compounds also desirably possess a reactive side chain, through which the molecule can be coupled to the support matrix.

When using additional adsorbant substrate in the practice o the present invention, the benefits of the invention are retaine independent of the order in which the adsorbant substrates are employed. While the experimental examples necessarily disclose certain order, it will be readily understood that no limitation is expressed or implied thereby.

In those embodiments wherein a second adsorbant substrate comprising lysine was employed, generally the t-PA solution containing approximately 1.0 M NaCl obtained from the Zn ++

chelate resin was diluted ten-fold with 25 mM Tris-HCl (pH 7.5),

0.1% Tween 80 or -Pluronic F-68 aήσTT.0 KIU aprotinin/ml and passe over L-lysine-Sepharose resin at 4° C at a rate of approximately two column volumes per hour. In these embodiments, it was discovered that diluting the t-PA with buffer containing 0.1% detergent resulted in greater recovery of t-PA than if the solution had been diluted with buffer containing only 0.01% of the detergent.

The binding efficiency of t-PA to the resin is in part dependent upon the temperature, pH and salt concentration of the medium to be contacted. The binding capacity of the resin was increased with decreasing temperature. The optimal binding of t

PA to the resin occurs at pH 7 to 8, and when the ionic strength of the medium is equivalent to approximately 100 mM NaCl.

Dilution, dialysis or gel filtration can be used to modify the ionic strength of the liquid medium to obtain the optimum benefits of the present invention. To ensure optimal binding of t-PA, approximately 1 liter of resin is used for each 0.2 g of t- PA. If conditioned tissue culture medium is directly contacted with the adsorbant, the optimal dilution is approximately one part medium to three parts 20 mM Tris-HCl, 0.1% Tween 80.

The lysine-Sepharose with bound t-PA was washed with a buffer, (20 mM Tris-HCl pH 7.5, 0.01% Tween 80, Pluronic F-68, or 0.05% Zwittergent 3-12 and 500 mM NaCl) to remove unrelated proteins, and thereafter the t-PA was eluted with this solution but at a pH greater than 8.5 or with the same solution containing an eluting agent such as 10-20 mM 6-amino-n-hexanoiσ acid, 20-50 mM L-lysine or 100-300 mM L-arginine.

While it is considered desirable to elute the tissue plasminogen activator by means of a competitive agent, it will be readily appreciated that other means can be used to elute the t- PA from the adsorbant substrate including, for example, alterations in pH, ionic strength of the buffer, and the addition of various chaotropic agents. However, it is considered desirable to use the least disruptive agent for the elution procedure in order to maximize the recovered plasminogen activator activity.

It is also considered desirable to use an elution procedure that will facilitate subsequent formulation of the t-PA for storage and therapeutic use. The lysine-Sepharose with bound t- PA is washed with a buffer at pH 7.5 consisting of 10 mM Tris, 500 mM NaCl and 0.01% Pluronic F-68 and followed by a buffer of pH 8 (for example, 3 mM Na glutamate containing 160 mM NaCl, 0.01% Pluronic F-68). The bound t-PA can then be eluted by washing the resin with a buffer of pH 4 (for example 3 mM Na- glutamate containing 160 mM NaCl and 0.01% Pluronic F-68). This solution containing the t-PA can be directly concentrated, for example by pressure dialysis using an Amiσon pressure dialysis cell with a YM30 membrane (Amicon) or with an analogus membrane in cross-flow apparatus. Using this system at pH 4 it is possible to concentrate t-PA to greater than 1 mg/ml. It is important that the pH be maintained relatively acidic to effect concentration. It has surprisingly been found that t-PA becomes insoluble at concentrations of 0.1 mg/ml or greater if the pH exceeds 5.

After concentration 5 mg/ml of mannitol can be added. This solution can be lyophilized and reconstituted by the addition of water without any loss of activity. In a buffer containing 0.1% Pluronic F-68, 160 mM NaCl and 3 mM Na glutamate (pH 4.0), the t-PA activity is stable for at least 7 days at 23° and indefinitely when frozen. The t-PA formulated in this manne was shown to actively mediate the dissolution of blood clots whe administered to rabbits and dogs.

Cation exchange chromatography can also be used to concentrate the t-PA. The t-PA eluted from the lysine solumn at pH 4 can be directly passed through a column of S-Sepharose-FF (Pharmcia, Inc. ) equilibrated at 4° with the same lysine column elution buffer. The t-PA is then eluted at pH 5.0 (3 mM Na- glutamate or 2.5 mM Na citrate, 0.01% Pluronic F-68 containing 200-500 mM NaCl.)

Non-ionic detergents are ordinarily used during cell extractions and chromatography to increase t-PA yields and reduce non-specific adsorption. The use of non-ionic detergent such as Tween 80 of Triton X-100 to enhance the recovery of t-PA is well known (Rijken et al. , supra) . However, since most common non- ionic detergents have critical micellar concentrations on the order of 0.001%, they can not be effectively removed by simple dialysis, and therefore impede the concentration of solutions. Zwittergent 3-12 works effectively in ensuring high yields of t- PA, and can be used at a concentration of 0.05%, less than one- half of its critical micellar concentration. Because of its relatively high critical micellar concentration, the detergent can be removed effectively by dialysis or gel filtration. The t- PA can thereby be formulated at the desired concentration with appropriate surfactants (for example Pluronic F-68) added back, if desired, at concentrations appropriate for intravenous use.

We have chosen to use Pluronic F-68 in our final t-PA formulations. It is a more effective detergent at pH 4-5, the optimal range for concentrating t-PA, than is Zwittergent 3-12. Pluronic F-68 also has significant advantages over Tween 80 which is widely used to stabilize t-PA (Rijken & Collen, supra) . It is less toxic than Tween 80 and can be lyophilized to a powder, therefore, making it more compatable in pharmaceutical formulations.

Compounds of the present invention, prepared as disclosed are shown to have the capability of recognizing and binding to fibrin, which is present in a host's circulatory system at locations of thromboses. These compounds are also shown to have fibrinolytic activity and, therefore display thrombolytiσ activity as well. Preparations of t-PA produced by the methods of the present invention are an improvement over t-PA prepared by other procedures in that the enzyme will be consistently and substantially pure one-chain, substantially free of degradation products and can be concentrated and formulated in solutions for therapeutic uses. The methods of the present invention will not result in the contamination of the product with elements of the chromatographic resins likely to be antigenic or tumorgenic. The absence of degradation products from these preparations provide a thrombolytic agent having greater specificity and less systemic activation of plasminogen.

Compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutical applications such as, e.g., dissolving blood clots. Thus, these compounds can find use as therapeutic agents in the treatment of various circulatory disorders, such as, for example, coronary or pulmonary embolism, stroke and decreased peripheral blood flow.

These compounds can be administered to mammals for veterinary use such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents, that is, in a physiologically acceptable carrier. In therapy dependent on t-PA, it may be important to achieve high plasma levels of t-PA very rapidly by injection. In such cases it will be necessary to have t-PA available in solutions of appropriate concentrations (1 to 10 mg/ml or greater) . Physiologically acceptable carriers or methods for maintaining t-PA in solution at concentrations in this range have not been known prior to the present invention. In general the administered dosage will range from about 0.01 to 100 mg/kg, and more usually 0.1 to 10 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.

These compounds can be administered neat, as mixtures with other physiologically acceptable active or inactive materials, or with physiologically suitable carriers such as, for example, water or normal saline. At the concentrations necessary for therapeutic administration it may be necessary to maintain t-PA w th an appropriate detergent in the compound to prevent aggregation of the protein. The compounds can be administered parenterally, for example, by injection. Injection can be subcutaneous, intravenous, or by intramuscular injection. These compounds are desirably administered in pharmaceutically effective amounts and often as pharmacologically acceptable salts such as acid addition salts. Such salts can include, e.g., hydrochloride, hydrobromide, phosphate, sulphate, acetate, benzoate, malate, among others.

Compounds of the present invention can also be used for preparing antisera for use in immunoassays employing labelled reagents, usually antibodies. These compounds and immunologiσ reagents may be labelled with a variety of labels such as chromophores, fluorophores such as, fluorescein or rhodamine, or radi .oi.sotopes such 125I, 35S, 14C, or 3H, or magnetized particles, by means well known in the art. These labelled compounds and reagents, or labelled reagents capable of recognizing and specifically binding to them, can find use as e.g., diagnostic reagents. Samples derived from biological specimens can be assayed for the presence or amount of substances having a common antigenic determinant with compounds of the present invention.

In addition, monoclonal antibodies can be prepared by methods known in the art, which antibodies can find therapeutic use, e.g., to neutralize overproduction of immunologically related compounds in vivo. In addition, the t-PA as prepared in this invention when suitably labelled with radioisotopes such as 131I, 123I, 111In or ^c may prove useful for the detection and localization of thrombi in patients (U.S. Patent Application No. 518,438) .

The following examples are provided by way of illustration, rather than implying any limitation of the subject invention.

Experimental

Example I :Purification of t-PA from Conditioned Liquid Medium

Liquid medium (1:1 mixture Ham's F-12 and DMEM) containing 0.5%- fetal bovine serum, which had been pre-adsorbed with lysine- Sepharose, and 10 KIU aprotinin per ml was conditioned by incubation with Bowes melanoma cells (Rijken and Collen, supra; Kluft et al, supra) , or alternatively, other plasminogen activator producing cells. This conditioned liquid medium was clarified by centrifugation at 10,000 x g for 30 minutes at 4°C or by filtration through low-protein binding membranes (e.g., Gel an Acrodisc 50A) or filter cartridges (e.g., Sartorius, type CA αr PH) . With the cartridge filters it is considered desirable to pretreat the membranes by pre-wetting with a solution of 0.1% Tween 80 or Pluronic F-68 to decrease the adsorption of t-PA to the membranes.

Clarified medium was adjusted to approximately pH 7.2 to 7.4 with NaOH, chilled to 4°C, and passed through a chelating Sepharose column complexed with Zn as recommended by the manufacturer (Pharmacia, Inc.). The column had been previously equilibrated with phosphate buffered saline. Up to 200 equivalent column volumes of medium were passed through the resin at rates up to 50 cm h for a 10 cm bed of Sepharose CL-6B or 300 cm h~ for an equivalent column of Sepharose-FF; and greater than 95% of the t-PA activity was bound to the resin. The column was washed at a rate of 50 cm h~ with 20 mM Tris-HCl, 1.0 M NaCl, 0.01% Tween 80, 10 KIU aprotinin per ml until the absorbance (280 nm) of the eluent buffer was equal to that of the applied buffer. The column was then washed with two to three column volumes of 20 mM Tris-HCl (pH 7.5), 25 mM NaCl, 0.01% Tween 80, 10 KIU aprotinin per ml. The t-PA activity associated with the degraded forms of the enzyme was eluted with 20 mM Tris- HCl (pH 7.5), 25 mM NaCl, 0.1 M imidazole, 0.01% Tween 80 (termed eluate "Zn A") . The intact enzyme was recovered by passing 20 m Tris-HCl (pH 7.5), 1.0 M NaCl, 50 mM Na EDTA, 0.01% Tween 80 through the resin (termed "Zn B") . The elution profile is shown in Figure 1A. Fractions, typically 1/4 column volume, were collected and aliquots assayed for t-PA activity using appropriate methods. The t-PA containing fractions of the "Zn B elution were collected, diluted ten-fold with cold 20 mM Tris-HC (pH 7.5), 0.1% Tween 80, 10 KIU aprotinin per ml and loaded at a rate of 25 cm per hour onto a 10 cm high bed of lysine-Sepharose A column was chosen such that approximately 1 liter of resin was available for each 0.2 g of t-PA.

The lysine-Sepharose was washed at 4°C with one column volume of 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 80, 10 KIU aprotinin per ml at a rate of about 25 cm per hour. The column was then washed with 20 mM Tris-HCl (pH 7.5) , 500 mM NaCl, 0.05% .Zwittergent 3-12, 10 KIU aprotinin per ml until the absorbance of the eluent buffer was equal to the applied buffer.

Bound plasminogen activator was eluted by washing the column with- 20 mM Tris-HCl, 500 mM NaCl, 50 mM L-lysine, 0.05% Zwittergent 3-12. Approximately two volume equivalents of elution buffer were required to complete the recovery (Figure IB) .

Alternatively, t-PA may be eluted from the column by lowering the pH. A second Zn -chelate Sepharose was loaded with serum-free conditioned medium and chromatographed as described above. The recovered intact t-PA (Zn B) was diluted and loaded onto a lysine-Sepharose column. This column was washed at 4°C with 10 mM Tris pH 8.0, 500 mM NaCl, 0.01% Pluronic F-68 until the adsαrbance of the eluent buffer was equal to the applied buffer. The lysine-Sepharose column was washed with 3-4 column volumes of 3 mM glutamic acid pH 8.0, 160 mM NaCl, 0.01% Pluronic F-68.

Bound plasminogen activator was eluted with 3 mM glutamic acid pH 4.0 160 mM NaCl, 0.01% Pluronic F-68. The t-PA concentration was 0.2 - 0.3 mg/ml and the pH of the eluent was 4.4 + 0.1 (Figure IC) .

This eluted t-PA was concentrated up to 1 mg/ml over an Amicon YM-30 membrane by pressure dialysis. To avoid unspecific binding of plasminogen activator, the membrane was pretreated with 3 mM glutamic acid pH 4.0, 160 mM NaCl, 0.01% Pluronic F-68.

After concentration, the t-PA solution was brought up to an Pluronic F-68 concentration of 0.1%. Five mg of mannitol was added per ml. This solution was lyophilized and reconstituted b the addition of water without any loss of activity.

Summaries of the purification a t-PA from serum-supplemente and serum-free media are presented in Tables 1 and 2, respectively. The recovered t-PA, when analyzed by gel electrophoresis under non-reducing conditions (Laemmli, supra) had an apparent molecular weight of about 66,000 daltons and represented greater than 95% of the total protein. (Figure 2) . This t-PA exists primarily as the one-chain form as evidenced by absence of 32,000 and 34,000 subunits of two-chain seen under reducing conditions (Figure 2) . The enzymatic, physiochemical and antigenic properties of the recovered protein confirmed that the material was tissue plasminogen activator. Amino acid sequencing (Applied Biosystems Model 470A Sequencer) indicated the presence of two molecular forms with N-terminal sequences shown in Table 3. The sequences and the N-terminal heterogeneit are as reported in the literature. (Wallen et al Eur. J. Biochem 132:681-686 (1983); Pohl et al. Biochemistry 23:3701-3707 (1984)) .

When the procedures described above are used, 5-25% of the total t-PA is in the form of degraded t-PA, and therefore recovered in the "Zn A" eluate. If liquid medium containing serum, which has not been pre-adsorbed with lysine-Sepharose was use, 25 to 100% of total enzyme eluted from the Zn-chelate column is found in the Zn A eluate. Zymographic analysis (Granelli- Piperno & Reich, supra) of typical "Zn A" and "Zn B" pools are shown in Figure 3 and demonstrated the separation of low molecular weight forms of t-PA from the bulk of the intact t-PA.

Table 1; Recovery of t-PA from serum-supplemented medium conditioned by Bowes melamoma cells. The lysine-Sepharose column was eluted at pH 8.0 as described in the text.

Volume Total Protein t-PA t- •PA Recovered Step (ml) (mg) (I.U.) (%)

Harvest 5500 1650 983,000 100

Zn -chelate

Zn A 32 52 37,000 4

ZN B 100 29 681,000 70

Lysine- Sepharose 28 1.3 739,000 75

Table Legend: Protein was determined relative to bovine serum albumin by the BCA method (Pierce Chemical Company.) t-PA activity was determined using zonal clearing on plasminogen- enriched fibrin plates (Haverkatet & Brakman, Prog. in. Chem. Fibrin. Thromb. 1 :151-159 (1975)) and was measured relative to a standardized preparation of t-PA, activity of which had been previously defined relative to the WHO International t-PA standard.

ACS/CODON/2/PAT13 Table 2: Recovery of t-PA from serum-free medium conditioned by Bowes melomona cells. The lysine-Sepharose column was eluted at pH 4.0 and the final t-PA solution was concentrated by ultralfilitration as described.

Total

Step Volume Protein t-PA Recovery (ml) (mg) (I.U) (%)

Harvest 43,400 1,910 9,766,000 100

Zn -chelate

Zn A 960 393 1,366,000 14

Zn B 230 46 7,931,000 81

Lysine-Sepharose 103 31 7,800,000 80 t-PA Concentrate 31 31 7,740,000 79

Table 3: Amino-terminal sequence of t-PA. Purified protein was subjected to automated sequence analysis on an Applied Biosystems Model 470A Protein Sequencer. At each cycle two amino acids present in a ratio of 3:2 were detected. These data yielded two sequences which differed by the presence or absence of three residues. (*) indicates the reported alternative amino terminus.

Cycle No. Major Peak Minor Peak Predicted Sequence

1 Gly Ser Gly

2 Ala Tyr Ala

3 Arg Gin Arg

4 Ser Val *Ser

5 Tyr He Tyr

6 Gin Cys Gin

7 Val Arg Val

8 He Asp He

9 - Glu Cys

10 Arg Lys Arg

11 Asp Thr Asp

12 Glu Gin Glu

13 Lys Met Lys

14 Thr He Thr

15 Gin Tyr Gin

16 Met Gin Met

17 He - He

18 Tyr His Tyr

19 Gin Gin Gin

20 - Ser Gin His Gin Ser

Example II: Removal of Degraded t-PA from Recovered t-PA

t-PA was recovered from conditioned media which had been supplemented with 0.5% serum. The conditioned medium was applie to a Zn ++-chelate Sepharose and the t-PA recovered using the - 38 -

protocol taught in the literature (Rijken et al, supra, Rijken & Collen, supra) . The recovered t-PA, containing intact and degraded enzyme, was then chromatographed on lysine- Sepharose as described above. This preparation of t-PA, which contained approximately equal amounts of intact and degraded t-PA, and which was contaminated with other unrelated proteins was dialyzed against 20 mM Tris-HCl (pH 8.5), 1.0 M NaCl, 0.01% Tween 80, or other buffers appropriate for the binding of t-PA to Zn-chelating Sepharose resin. This material (Figure 4, "Load") was applied to a column of the resin, and washed and eluted as described in Example I. As expected the majority of degraded t- PA was eluted from the column in the "Zn A" fraction (Figure 4) whereas the intact t-PA now substantially free of the 50,000 mw form was recovered in the "Zn B" fraction (Figure 4) .

Example III: Recovery of t-PA from-E. coli Extracts

A guanidine-HCl extract of E. coli expressing pre-pro-t-PA was prepared as described previously (Pennica et al. , Nature 301:214-221 (1983)). The extract was diluted to a concentration of 1 M in guanidine-HCl with 20 mM Tris-HCl (pH 7.5), 0.01 M NaCl, 0.01% Tween 80 and loaded onto the Zn⁺⁺-chelate Sepharose column. Chromatography on the Zn -chelate and lysine Sepharose columns proceeded as described in Example I with the intact E. coli t-PA activity eluting as expected for intact mammalian cell enzyme.

Example IV: Effect of pH on the solubility of t-PA

Aliquots of a solution of intact t-PA at a concentration of approximately 0.1 mg/ml were dialyzed to equilibrium against 10 mM buffers of several pH values containing 160 mM NaCl and 0.1% Tween 80. Each sample was transferred to a centrifuge tube, mixed thoroughly and an aliquot was assayed on a plasminogen enriched fibrin plate as described in Table 1. The sample was centrifuged at 16,000 g to sediment insoluble material. The t-PA activity remaining in the supernatant fractions was assayed on fibrin plates. Prior to centrifugation each sample was shown to contain the same amount of t-PA; however, in those samples with pH values greater than pH 5 and up to at least pH 10.5 a substantial fraction of the t-PA was contained in aggregates which could be removed by centrifugation (Table 4) .

Table 4: Solubility of t-PA at various pH value. The t-PA remaining in solution after centrifugation of the samples was determined on fibrin plates. All samples prior to centrifugation contained approximately 46,000 units/ml.

Soluble t-PA 25 (I.U./ml)

4.0 46,000

5.0 46,000

6.0 34,000

7.8 8,500 9.3 15,500

10.5 34,000

This experiment demonstrates that at neutral pH values t-PA aggregates even in relatively dilute solutions. Therefore, to concentrate t-PA for use in a pharmaceutical formulation weakly buffered solutions of acidic pH should be employed.

Example V: The use of cation exchange chromatography for the concentration of t-PA.

The experiments described in Example IV demonstrate that t- PA is maximally soluble at acidic pH. The isoelectric point of t-PA is approximately pH 7.5 to 8, therefore in acidic solutions t-PA should possess a net positive charge and bind to cation exchange resins such as SP-Sepharose or S-Sepharose-FF. These cation exchangers typically will reversibly bind 10 to 100 mg of protein per ml of resin, and thus provide a matrix for the concentration of t-PA.

One ml of S-Sepharose Fast Flow was equilibrated with 0.01 M sodium acetate, 150 mM NaCl, 0.01% Tween 80, 0.02% sodium azide at pH 4.5 and then packed into a 0.5 cm (i.d.) column. Five mg of t-PA in 150 ml of 3 mM glutamic acid, 160 mM NaCl, 0.01% Pluronic F-68, pH 4.0 was applied to the resin at approximately 50 ml h^*"1.

The column was washed at 12 ml h~ with 2.5 mM sodium citrate, 100 mM NaCl, 0.1% Pluronic F-68, pH 5.0 until the adsorbancy at 280 nm of the effluent equaled that of the solution applied to the column. The column was eluted at 12 ml hr ~ with 2.5 mM sodium citrate, 1 M NaCl, 0.07% Pluronic F-68 at pH 5.0. Fractions, typically 1/4 column volume were collected and aliquots were assayed for t-PA activity (Table 5) .

Table 5: Recovery of t-PA from S-Sepharose

Fast Flow. t-PA activity was assayed as described Table 1.

Sample Volume t-PA Recovery (ml) (I.U.) (%)

t-PA load 150 3,000,000 100

S-Sepharose FF

Flow through 5 500 0

Peak fractions 8.5 3,500,000 115

The product was concentrated by a factor of 20 to a final concentration of 0.65 mg/ml with full recovery of activity. Thi t-PA solution was dialyzed to equilbrium without loss of activit against a solution containing 3 mM glutamic acid, 160 mM NaCl an 0.01% Pluronic F-68 (pH 4.0). This solution is suitable for further concentration by ultrafiltration or direct formulation i a pharmaceutical preparation. Example VI: Comparison of Adsorbant Substrates

Chromatographiσ resins were synthesized by dissolving approximately one millimole of each of several diaminocarboxylic acids in one ml of 0.1 M sodium bicarbonate. Each acid was added tα 3 ml of a 66% slurry of CNBr-activated Sepharose or activated CH-Sepharose in water. Solutions were mixed with gentle agitation for 20 minutes at 4 C.

The coupling reactions were terminated by the addition of 200 ml of triethanolamine. After an additional 30 minutes of agitation at 4°C, the substrates were washed as suggested by the manufacturer of the Sepharose.

One-half of each packed resin was transferred to a small column and washed with 5 ml of 20 mM Tris-HCl, pH 8.0, 0.1% Tween 80. t-PA samples containing 2000 units in 5 ml of the same buffer were passed over each column. Each adsorbant substrate was washed with 5 ml of the same buffer. Thereafter, t-PA was eluted with 20 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 0.2 M e- a inocaprioic acid, 0.1% Tween 80. The enzyme activity recovered thereby was measured on plasminogen enriched fibrin plates (Haverkatet & Brakman, supra) to calculate the fraction of enzyme bound by the adsorbant. The results, as shown in Table 6 below, demonstrated that L-lysine provides the best chromatographic ligand and that a six carbon spacer between the solid support and the ligand improved the efficency of t-PA binding. Table 6: Binding of t-PA to Immobilized Diaminocarboxylic Acids t-PA Units Bound

Immobilized Ligand CH-Sepharose Sepharose

2,3-diaminopropionic acid 11 4

D,L-orinithine 16 4

D-lysine 360 64

L-lysine 1040 780

2,4-diaminobutyric acid 80 56 diaminopimelic acid 180 4

Example VII: The Effect of Aprotinin on Yield of One-Chain t-PA from various tissue culture media

Aprotinin is known to inhibit the conversion of one-chain t- PA into two-chain t-PA. (Rijken & Collen, supra) The concentration of aprotinin necessary to optimize recovery of one- chain t-PA relative to two-chain degraded forms of t-PA was determined. A genetically engineered strain of Bowes melanoma cells was grown to confluency in a 24 well plate in a medium composed of a 1:1 mixture of Ham's F-12 and DMEM (F-12/DMEM) supplemented with 5% heat-inactivated fetal bovine serum. The growth medium was removed, and the cells were washed with serum-fre F-12/DMEM. Serum-free medium, medium supplemented with 0.5% heat-inactivated fetal bovine serum which had been pre-treated with lysine-Sepharose, or medium supplemented with 0.5% heat- activated fetal bovine serum was added to the cells. Aprotinin was added to each of the media so that individual wells in the tissue culture plates contained 0, 1, 5, 10, 50 or 100 KIU of aprotinin/ml. The plates were incubated at 37° C for 48 hours.

The media were harvested, clarified by centrifugation and assayed for t-PA activities. The total t-PA in each sample was determined from the diameter of the zone of clearing effected by a.5 ul sample placed into a well formed in a plasminogen enriched fibrin plate (Haverkatet and Brakman, supra) . Neither the choice of medium nor concentration of aprotinin had any effect on total tr-P production. Each sample contained approximately 900 I.U. t- PA per milliliter.

The effect of aprotinin and medium on the conversion of one- chain t-PA to the two-chain form was determined by Western blot analysis (Burnette, Anal. Biochim. 112:195 (1981)). The protein from one ml of each sample of the conditioned media was recovered by precipitation with trichloroacetic acid (10% final concentration) . The pellet of protein obtained by centrifuging the samples for 10 minutes at 15,000 g was resolubilized in 20 ul of sample buffer (Laemmli, supra) . The samples, which contained 10 mM dithiothreitol, were boiled for 7 minutes; then loaded onto a 8.75% polyaσrylamide gel. After running the dye front to the bottom, the proteins were electroblotted onto nitrocellulose.

The nitrocellulose was incubated in 5% BSA in 10 mM Tris- HCl pH 7.5, 0.9% NaCl for 30 minutes at room temperature, and the incubated with rabbit anti-t-PA (antiserum to denatured human t- PA) (10 microliters serum in 10 ml of 10 mM Tris-HCl pH 7.5, 0.9% The nitrocellulose was incubated in 5% BSA in 10 mM Tris- HCl pH 7.5, 0.9% NaCl for 30 minutes at room temperature, and the incubated with rabbit anti-t-PA (antiserum to denatured human t- PA) (10 microliters serum in 10 ml of 10 mM Tris-HCl pH 7.5, 0.9% NaCl 3% BSA, 0.05% Tween 20) overnight at 4°C. The binding of the rabbit anti-t-PA was detected using the Vectastain ABC (avidin-biotin-horseradish peroxidase complex) kit and 4-chloro-l naptithol as the substrate for the peroxidase (Figure 5) . On the blots, one-chain t-PA is seen as a band at approximately 66,000 mw, while the subunits of the two-chain enzyme are detected as bands at 32,000 and 34,000 mw. In those experiments wherein serum was used no single-chain t-PA can be visualized. In control experiments this was shown to be the result of the large amount of albumin in the sample which both distorts the single- chain t-PA band during the gel electrophoresis and further inhibits the complete binding of proteins in this molecular weight range to the nitrocellulose. However, the presence of serum had no effect on the migration of transfer of the two- chain bands.

Complete inhibition of the conversion of one-chain t-PA to two-chain was observed at 5-10 KlU/ml aprotinin for either serum- free medium (Figure 5) or medium containing 0.5% "scrubbed serum" (Figure 5C) . When "non-scrubbed" serum was used even 100 KIU aprotinin per ml was not adequate to completely inhibit formation In media containing serum the conversion of one-chain t-PA to:,two-chain t-PA is a result of proteolytic activities involving t-PA as a substrate. These activities will additionally cause degradation of t-PA. Eliminating or blocking the activity which causes degradation of t-PA is an important step in maintaining the= integrity of t-PA in its medium.

Example VIII: The stimulation of "intact" and

"degraded" t-PA by fibrinogen fragments.

A striking difference between tissue plasminogen activators and urokinase is that the former adsorb to fibrin (Thorsen et al supra) _r which results in a marked enhancement of the activation of plasminogen (Wallen, Prog. Chem. Fibrinolysis Thrombolysis 3_:1.67-181 (1978)). Fragments generated from a CNBr cleavage of fibrinogen (Niewenhuizen et al, Biochim. Biophys. Acta. 755:531- 533 (1983)) also stimulate the process of plasminogen activation by t-PA.

Twenty 1 of t-PA solutions each containing 0.2 units of one-chain, two-chain or degraded (50,000 mw) t-PA as measured by fibrin, plate assay, was added to 180 1 of solution containing plasminogen, the chromogenic substrate S-2251 (Kabi) , and with or without fibrinogen fragments (Niewenhuizen et al, supra) . In thi assay (Wiman et al, Biochim. Biophys. Acta 579:142-154 (1979)) t- PA cleaves plasminogen to form active plasmin. The resulting plasmin activity is assayed using the chromogenic substrate S- 2251, which yields a yellow color (A₄₀₅ ) , upon hydrolysis by plasmin. The mixture (0.2 units t-PA, 0.2 mM S-2251, 20 Λg/ml CNBr-fragments of human fibrinogen) was incubated at 37° and the absorbance change at 405 nm was read at 15 minute intervals. Activity is determined from a plot of adsorbace vs (time) which is linear (Drapier et al, Biochimie 61:463-571 (1979)).

As is shown is Table 7 equivalent amounts of t-PA, as defined by equal zones of clearing on a fibrin plate, exhibited very different activities in the S-2251 assay. In the absence o fibrinogen fragments two-chain t-PA was much more active than either one-chain or degraded t-PA. The addition of fibrinogen fragments greatly stimulated the activity of the one-chain enzym and to a lesser extent that of the two-chain form such that the resulting activities were equivalent. The degraded (50,000 mw) t-PA was much less stimulated by the fibrinogen fragments.

These data suggest that the best form of t-PA for therapeutic applications is the one-chain enzyme as its activity is much more fibrin-specific than that of the two-chain form. Both forms of the intact enzyme are much more fibrin-specific than the degraded t-PA.

Table 7: Stimulation of t-PA by fibrinogen fragments(FF)

Enzyme Activity ( A.../mm . 2 x 105) Fold Stimulation

-FF +FF

One-chain 0.06 14.8 250

Two-chain 0.34 16.3 48

Degraded 0.08 1.5 19 (50,000 mw) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarit of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. However, it must be stressed that the production of intact t-PA which is suitable for subsequent formulation in pharmaceutical compositions requires following the essential steps described in the foregoing invention.

Claims (46)

1. A method for recovering intact tissue plasminogen activator (t-PA) from a liquid medium containing said t-PA comprising contacting said liquid medium containing intact t-PA and degraded t-PA with at least one adsorbant substrate capable of adsorbing the intact and degraded t-PA and separately eluting the adsorbed degraded t-PA from the substrate and the intact t-P from the substrate.

2. The method as recited in Claim 1 wherein said liquid medium comprises a medium conditioned by incubation with t-PA producing cells.

3. The method as recited in Claim 2 wherein said t-PA producing cells comprise eukaryotic cells.

4. The method as recited in Claim 3 wherein said t-PA producing cells comprise mammalian cells.

5. The method as recited in Claim 4 wherein said t-PA producing cells comprise melanoma cells.

6. The method as recited in Claim 5 wherein said t-PA producing cells comprise Bowes melanoma cells.

7. The method as recited in Claim 2 wherein said t-PA producing cells comprise bacterial cells genetically engineered to contain a gene encoding for t-PA.

8. The method as recited in Claim 2 wherein said conditioned liquid medium comprises at least one medium selecte from the group consisting of serum-free medium, serum- suplemented medium, serum-fraction supplemented medium and albumin-supplemented medium.

9. The method as recited in Claim 8 further comprising pre-treating said serum or serum fraction medium prior to the incubation with said t-PA producing cells with an additional adsorbant substrate capable of removing substantially all plasminogen present in the serum or serum-fraction medium.

10. The method as recited in Claim 2 wherein said liquid medium further comprises aprotinin during said conditioning incubation.

11. A method of recovering intact tissue plasminogen activator (t-PA) from a liquid medium containing said intact t-P and at least one of degraded t-PA and other unrelated proteins comprising the steps of: a) contacting said liquid medium with a metal chelate adsorbant substrate selected from divalent cation chelates; b) subjecting said metal chelate adsorbant substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact t-PA; and c) subjecting said metal chelate adsorbant of step b to at least one second solution which selectively dissociates therefrom the intact t-PA.

12. The method as recited in Claim 11 wherein said first solution comprises a low ionic strength solution having a salt concentration in the range of 25 millimolar to 100 millimolar an said second solution comprises a high ionic strength solution having a salt concentration in the range of 100 millimolar to 4 molar.

13. The method as recited in Claim 12 wherein said salt is sodium chloride.

14. The method as recited in Claim 13 wherein said first solution comprises 25 mM sodium chloride and said second solutio comprises 1 molar sodium chloride.

15. The method as recited in Claim 11 wherein said first solution further comprises at least one first disrupting agent capable of selectively disrupting the interaction between said degraded t-PA and said metal chelate adsorbant.

16. The method as recited in Claim 11 wherein said second solution further comprises at least one second disrupting agent capable of selectively disrupting the interaction between said intact t-PA and said metal chelate adsorbant.

17. The method as recited in Claim 15 wherein said first disrupting agent comprises 25-250 mM imidazole.

18. The method as recited in Claim 15 wherein said first solution comprises 25 M sodium chloride and 100 mM imidazole.

19. The method as recited in Claim 16 wherein said second disrupting agent is selected from imidazole, zinc, sodium ethylenediaminetetraacetiσ acid and derivatives thereof.

20. The method as recited in Claim 19 wherein said second disrupting agent comprises 10-250 mM sodium ethylenediamine- tetraacetiσ acid.

21. The method as recited in Claim 16 wherein said second solution comprises 1 molar sodium chloride and 50 mM sodium ethylenediaminetetraacetic acid.

22. The method as recited in Claim 11 wherein said metal chelate adsorbant substrate comprises molecules having the general formula:

CH.-COO support substrate - (CH_) - N Metal ++

CH₂-COO where n is greater than or equal to zero.

23. The method as recited in Claim 22 wherein said metal is zinc.

24. The method as recited in Claim 11 further comprising concentrating said intact t-PA.

25. The method of Claim 24 wherein said intact t-PA is concentrated in the presence of non-ionic or zwitterionic detergent.

26. The method of Claim 25 wherein said intact t-PA is concentrated by lyophilization in the presence of at least one stabilizing agent.

27. The method of Claim 26 wherein said stabilizing agent is mannitol.

28. A method of recovering intact tissue plasminogen activator (t-PA) from a liquid medium containing said intact t-P and at least one of degraded t-PA or other unrelated proteins comprising the steps of: a) contacting said liquid medium with a substrate comprising an immobilized arainocarboxylic acid; b) subjecting said immobilized aminocarboxylic acid substrate to at least one third solution that dissociates from said substrate degraded t-PA but not said intact t-PA; c) subjecting said immobilized aminocarboxylic acid substrate of step b to at least one fourth solution that dissociates from said substrate said intact t-PA.

29. The method as recited in Claim 28 wherein said aminocarboxylic acid is L-lysine.

30. The method as recited in Claim 28 wherein said third solution comprises a neutral pH solution and said fourth solutio is selected from solutions having a pH greater that 8.5, solutions having a pH less than 4.5 and solutions comprising at least one aminocarboxylic acid.

31. The method as recited in Claim 30 wherein said solutions having a pH of less than 4.5 are selected from citric acid and amino acid solutions having a pH of 4.0 or less.

32. The method as recited in Claim 30 wherein said third solution comprises 0.5 molar sodium chloride having a pH in the range of pH 5.0 to 8.0 and said fourth solution comprises 160 millimolar sodium chloride and 50 millimolar lysine.

33. The method as recited in Claim 28 further comprising concentrating said intact t-PA.

34. The method of Claim 33 wherein said intact t-PA is concentrated in the presence of non-ionic or zwitterionic detergent.

35. The method of Claim 34 wherein said intact t-PA is concentrated by lyophilization in the presence of at least one stabilizing agent.

36. The method of Claim 35 wherein said stabilizing agent is mannitol.

37. A method for recovering intact tissue plasminogen activcator (t-PA) comprising the steps of: a) providing a liquid medium selected from serum- free medium, serum-supplemented medium, serum-fraction supplemented medium and albumin-supplemented medium; b) pretreating said serum-supplemented or serum fraction supplemented medium with a first adsorbant substrate capable of removing substantially all plasminogen present in the serum-supplemented or serum- fractions supplemented medium; σ) adding to said liquid medium a plasminogen inhibitor; d) contacting said liquid medium with a metal chelate adsorbant substrate selected from divalent cation chelates; e) subjecting said metal chelate adsorbant substrate to a first solution which selectively dissociates therefrom degraded t-PA but not said intact t-PA; f) subjecting said metal chelate adsorbant of step e to at least one second solution which selectively dissociates therefrom the intact t-PA; g) contacting said liquid medium with a substrate comprising an immobilized aminocarboxylic acid; h) subjecting said immobilized aminocarboxylic acid substrate to at least one third solution that dissociates from said substrate degraded t-PA but not said intact t-PA; and i) subjecting said immobilized aminocarboxylic acid substrate of step h to at least one fourth solution that dissociates from said substrate said intact t-PA.

3.8. The method as recited in Claim 37 further comprising concentrating said intact t-PA.

39. The method of Claim 38 wherein said intact t-PA is concentrated in the presence of non-ionic or zwitterionic detergent.

40. The method of Claim 39 wherein said intact t-PA is concentrated by lyophilization in the presence of at least one stabilizing agent.

41. The method of Claim 40 wherein said stabilizing agent isrmaπnitol.

42. A biologically active compound of any of Claims 1-41 comprising intact t-PA substantially free from degraded t-PA..

43. A biologically active compound having thrombolytic activity comprising intact t-PA substantially free from degraded t-PA other proteins and peptides.

44. A biologically active compound as recited in Claims 42 or 43i further comprising intact one-chain t-PA substantially free from two-chain t-PA and degraded t-PA.

45. A pharmaceutical composition comprising a therapeutically effective amount of the compound of Claims 42 or 43 together with a physiologicaly acceptable carrier.

46. A method of treating a host in need of thrombolytic therapy comprising adminstering to said host an effective amount of the. composition of any of Claims 42 or 43.