CA1333163C - Methods for the recovery of tissue plasminogen activator - Google Patents

Abstract

A method for recovering t-PA from a liquid medium is disclosed. The method comprises contacting a liquid medium with at least one substrate capable of effecting a separation of intact t-PA from degraded t-PA thereafter recovering the intact t-PA free from other unrelated protein. The present invention also provides compounds produced by this method, compounds comprising intact one-chain t-PA and pharmaceutical compositions containing them and methods for using such compositions.

Description

1 ThiB ln~ention relate~ to the recov-ry of ti~su- plasminogen activator (t-PA) from liguid media and more ~pecifically, to an improved method for recovering intact single-chain t-PA substan-tially free of degraded t-PA and other non-homologous proteins.

Plasminogen activator~ have received attention for their role in the fibrinolytic system. These enzyme~ catalyze the conversion of the proenzyme plasminogen into the proteolytic enzyme plasmin; plasmin can, in turn, degrade fibrin, a ma~or 10 component of blood clots. Thus, plasminogen activators are potentially useful for the therapeutic treatment of blood clot~.
The known plasminogen activator~ include streptokinAFe, which is of bacterial origin, urokinase (u-PA), which has been i601ated from urine and culture fluids, and tissue plasminogen activator (t-PA), which is now becoming available from cultured human cell~ (Rifkin et al., J. Exp. Med. 139:1317-1328 (1974);
Wilson et al, CAncer Res. 40:933-938 (1980)). Streptokinase and U-PA are available commerci~lly, but appear not to posses~ the therapeutic efficacy of t-PA.

la ;

1 Intact t-PA ~8 a gly~o~I~Lein 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 eukaryotic cells grown in the presence of drugs that prevent glycosylation lo (Little et al., Biochemistry 23:6991-699S (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 t-PA (Thorsen et al., Throm. Diath. Haemorrh. 28:65-74 (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 ~ett. 163:37-41 (1983)), do not appear to display the specificity and clot localization properties ':

~ 333 ~ 63 1 of intact one-chain and two-chain t-PA. Further, it is believed that single-chain t-PA ic 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.
57:407-412 (1984)).
Various protocols have been described for the purification of t-PA using chromatographic, electrophoretic, and selective extraction and precipitation methods. Most of these methods, lo 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 132:681-686 (1983); Nielsen et al, EMB0 J. _: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 (:

1 forms of t-PA in preparations of the purified enzyme remains problematic to those skilled in the art (Rruithof 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, i6 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, gupra, 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 6erum containing production medium is used (Reagen et al, 6upra; 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).

The pre6ent lnvention provldes a rapid, efficient method for the recovery of intact, ~ingle-chain ti6sue pla6minogen activator6 (t-PA) from liguid media, e.g., 6erum-free and serum-6upplemented media u6ed to culture cell6 which secrete intact t-PA or from extract6 of cell6 which intracellularly deposit t-PA or non-glycosylated t-PA polypeptide. The novel method of the present invention effect6 the recovery of t-PA 6ubstantially free of degraded t-PA by contacting a liquid medium with at least one substrate capable of effecting a 6eparation of intact t-PA from degraded t-PA.
The present invention also provides method6 for further adsorbing t-PA onto additional adsorbant 6ubstrate6, e.g.
adsorbant 6ubstrates comprising at least one aminocarboxylic acid, followed by eluting and recovering the t-PA. Such additional ad60rption and elution can precede or follow the novel method6, whlle retaining the benefit6 of the present invention.
The present invention al60 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 6ubstrate 6uch as, e.g., lysine-Sepha-rosetm (Pharmacia Fine Chemical6, Piscataway, N.J.) chromatography.

, - 5a -1 The present invention further provides a method for recovering intact tissue plasminogen activator (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; c) 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 immoblized 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.
The present invention further provides a method of pretreating serum or fractionated serum for a medium in - 5b - 1 333 1 63 1 which cultured cells can be grown for the production of a protein, comprising contacting the serum or fractionated serum with an insoluble matrix containing pendant lysine groups, whereby substances selected from the group comprising proteolytic substances and other substances having affinity for lysine are removed from the serum or fractionated serum.
The present invention further provides a method of producing a protein in cultured cells grown in a medium containing serum or fractionated serum, comprising pretreating the serum or fractionated serum by contacting the serum or fractionated serum with an insoluble matrix containing pendant lysine groups, whereby substances selected from the group comprising proteolytic substances and other substances having affinity for lysine are removed from the serum or fractionated serum.
Untreated serum used in growth media for culture cells contains plasminogen and plasmin which are known to 1 proteolytically cleave t-PA (Wallen et al, 6upra). Lysine-Sepharose chromatography has been 6hown to be effective in the removal of these proteins from 6erum (Wu et al, Exp. Cell Research 96:37-46 (1975) Quigley et al J. Biol. Chem. Vol. 249, pg. 4306-4311 tl974)). Such depleted 6erum is capable of supporting the growth of tissue culture cells (Wu et al, 6upra;
Kaufman et al, Nolec. Cellular Biology 5:1750-1759 (1985)). The present invention provides improved methods for the removal of plasminogen and plasmin from 6erum, and further provides a novel use of "scrubbed 6erum" 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 6upplemented media.
Also provided are compounds and compositions obtained by practicing the present invention, 6aid compounds and compositions comprising intact t-PA, 6ubstantially 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 (Rijken et al., supra.) However, the prior art protocols differ 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 1 3~3 1 63 1 separating the bulk of the contaminating proteins, as well a8 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 adsorption effects. (Riiken et al, supra; Porath et al, Nature 258:598-599 (1975)). The present invention includes the unexpected observation that the use of a low ionic strength washing condition (under 100 mM salt, and preferably NaCl) allows for the elution of degraded lo 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.
Biochem. Biophys. 189:185-194 (1978)), cadaveric perfusates (Allen and Pepper, Thrombos. Haemostas. 45:43-50 (1981), and from medium conditioned by incubation with a guinea pig tumor cell line (Oerstein et al., Cancer Res. 43:1783-1789)). This substrate, however, has been reported ineffective for the purification of t-PA
found in human uterine tissue (Rijken et al., supra).

1 33s 1 63 1 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 admini~tration. 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 lo 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 3-12tm (Calbiochem, La Jolla, California), can be removed from t-PA
by dialysis or diafiltration. Alternatively, Pluronictm 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 ob;ect 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 (, . ~

1 in the culture of eukaryotic or bacterial cells, or from extracts of 6uch 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.
lo It is a yet another ob;ect 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 ob;ect of the present invention to provide a method for formulating t-PA in a concentration ~uitable 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.

l BRIEF DESCRIPTION OF THE DRAWINGS
Figure l 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 Sepharosetm. This column was developed as described in the text of Example l. 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-2288tm (Kabi~. The change in adsorbancy at 405 nm was monitored to measure the amidolytic activity of t-PA. The t-PA contained in the "Zn B" fractions was applied to a lysine-Sephaosetm column, and eluted either at pH 8 (Figure B) or at pH 4.0 (Figure C) as described in the text of Example l. 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 l from conditioned medium supplemented with pretreated serum. The left most lane contains a mixture of reduced and alkylated standard proteins, from top to bottom:
phosphorylase b (94,000 mw), albumin (67,000 mw), ovalbumin 1 (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 6hows 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 4C through a 0.75 mm thick 8.7% SDS
lo polyacrylamide gel using the Hoeffer "Mighty Smalltm"
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 c~r~

1 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 6eparation 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 6hows the inhibition by aprotinin of the conversion of one-chain to two-chain t-PA in various tissue culture media.
lo 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. _ 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 liguid 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 ( ~ 37J ~ 1 63 1 of effecting a separation of the intact t-PA from other unrelated proteins.
The pre6ent 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.

1 Such liquid media will generally contain a mixture of intact t-PA 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 a6 the 50,000 and 32,000 species.
Also included are those forms of t-PA which have been modified to alter their fibrin b1n~in~ or fibrin activation characteristic6, resulting in decreased thrombolytic activity or decreased 6pecificity.
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:

CH -COOH
~ upport- (CH2)n ~ N
substrate ~``~CH2-COOH
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, ~uch as lysine and propylsulfonate to further separate intact t-PA from undesirable contaminants.
For ease of use, the ligands effecting 6eparations are generally immobilized on support substrates. These support 1 substrates can compri~e any ~upport materials known to the art which do not interfere with the ~eparations as disclosed herein.
Such support 6ubstrates can be linked, e.g., convalently bound, to the 6eparation 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 6ubstrate, the term resin is used.
lo 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 freguently reguire 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-Williams and Porter, supra; Kluft et al, supra) and observed by us that the presence of ~erum 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 ~f the present invention where serum or fractionated ~erum was used to formulate the liquid medium, it was generally pretreated by adsorption to lysine-Sepharose. This pre-adsorbed serum ~upported survival and growth of the cell cultures equivalent to untreated serum (Wu et al, supra; Kaufman et al, supra.) This pre-treatment removed ~;ub-stantially all the plasminogen or plasmin from the serum (Deutsch and Mertz, Science 170:1095-1096 (1970). Plasminogen, when converted to plasmin by plasminogen activators, i8 known to lo 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 that 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 of 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 4C through a column of 1 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 the liquid medium. Approximately one milliliter of resin was used to treat each milliliter equivalent of undiluted serum. The level of plasminogen in sera varies 6ignificantly. It therefore is sometimes necessary to use amounts of resin greater than that specified above. With all ~erum tested, it was found that less lo resin was required for the complete removal of plasminogen if the serum is diluted as described here, than if undiluted 6erum is used as described in the literature (Wu et al, supra). It may be necessary with the diluted serum to adjust the osmotic ~trength 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 N NaCl, 50 mM Na EDTA (pH 7.5), followed by sterile water. The resin column was ~anitized by washing with 20% ethanol and then storing the column with ethanol for at least 18 hours. The resin was thoroughly washed with sterile water before re-use.
To further exemplify a presently preferred embodiment of one aspect of the present invention, 80wes melanoma cells, adsorbed to tissue culture flasks (Rijken and Collen, 6upra) or microcarriers tKluft et al., ~upra) were used to condition liquid 1 media which contained O to 0.5% scrubbed 6erum. Aprotinin at a concentration of 5 to lO0 RIU/ml, And typically 10 RIU/ml was included ~n 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-treat the filters by passing a solution of 0.1% Pluronic F-68tm (BASF) or Tweentm 80 (Atlas Chemical Company, Inc.) therethrough.
These conditioned media were chilled to approximately 4C, adjusted to between approximately pH 7 and 8 with 1 M HCl or NaOH, supplemented with 0.01% (w/v) Tween 80 or Pluronic F-68 and passed through a first column comprising Zn++ Chelate Sepharosetm or Zn++ Chelate Fast Flowtm 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 ~trength nor addition of 1 compounds toxic to ti6sue culture cells, so that the medium may be recycled into the cuiture, thus reducing the production costs related to media use. Optimal binding and recovery of t-PA was achieved when chromatography was performed at 4C using buffers of approximately pH 7-8, e.g., 20 mM Tris-HCl (pH 7.5 measured at 20C), 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 mM
NaCl, and desirably below 25 mM NaCl, is an important feature of the embodiments of the present invention employing metal chelate adsorbant ~ubstrates 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 ~trength of 25 mM NaCl, degraded forms of t-PA and the bulk o~ the non-related proteins adsorbed to the resin can be eluted as described below without significant elution of the desired intact form of t-PA.

1 Plasminogen activator6 which have been adsorbed during the practice of the present invention can be eluted from the 6ubstrate. When employing an adsorbant 6ubstrate, 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 6ites on the adsorbant.
For example, t-PA adsorbed to an adsorbant 6ubstrate comprising a metal chelate 6uch as zinc chelate can be eluted with imidazole, histidine or zinc, among others. Elution can also be effected by lo 6uch means as 6alt 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 selective 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 zymography (Granelli-Piperino & Reich, supra), using plasminogen-containing fibrin indicator plates, were eluted by washing the resin with 2S 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 immunoprecipitated by monoclonal antibodies directed against t-PA and therefore appear to be degraded t-PA.
u-PA i6 al60 eluted from the resin by this procedure. Since many tissue culture cells secrete u-PA, this chromatography procedure 1 3~3 1 63 1 ensures the recovery of t-PA free from this plaminogen activator which possesses less fibrin-clot 6pecificity.
The decreased ionic 6trength of the a~ueous phase of the intermediate washes, generally less than the equivalent of 100 mM
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 teaches the use of high ionic strength solutions to minimize non-specific ionic interactions of proteins with metal chelating resins. We lo 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 mM
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 i8 possible had the method for chromatography of t-PA on Zn++-chelate resin in the prior art been used (Rijken 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, 50 mM Na EDTA. Alternatively, the eluting buffer can contain 1.0 M
NaCl, 100 mM imidazole or gradually increasing amounts of NaCl 1 (0.025 to 1.0 M NaCl) with 100 ~M imidazole. The latter result6 in the cuccessive elution of t-PA ~ubpopulation6, di6tingui6hed 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+l-chelate resin can be further treated to remove additional, unrelated contaminants.
For example, the intact t-PA recovered from the Zn+l-chelate resin was then passed though a 6econd column comprising ~n aminocarboxylic acid (e.g., lysine) linked directly or via 6pacer (e.g. a 8iX carbon aliphatic 6pacer) to a 6upport substrate (e.g. Sepharo6e*). However, certain benefit~ of the present invention can be obtained with any compound wherein both an amino and carboxyl 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-hexanoic acid, among others. Included also are cyclic compounds such as tranexamic acid, and other analogs of lysine, such ~s 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 6upport matrix.
When usinq additional adsorbant ~ubstrate in the practice of the present invention, the benefits of the invention are retained *Trade mark 1 independent of the order in which the adsorbant substrates are employed. While the experimental examples necessarily disclose a certain order, it will be readily understood that no limitation is expreased or implied thereby.
In those embodiments wherein a ~econd 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 and 10 KIU aprotinin/ml and passed lo 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 ( !

f`3331 63 1 t-PA, approximately 1 liter of resin is used for each 0.2 g of t-PA. If conditioned ti6sue culture medium i6 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 O.OS% 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 ~uch as 10-20 mM 6-amino-n-hexanoic 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 :

1 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 m~ Na-~#'~ glutamate containing 160 mM NaCl and 0.01% Pluronic F-68). Thi6 solution containing the t-PA can be directly concentrated, for example by pressure dialysis using an Amicon pressure dialysis cell with a YM30 membrane (Amicon) or with an analogus membrane in cross-flow apparatus. Using this system at pH 4 it i8 possible to concentrate t-PA to greater than 1 mg/ml. It is lo 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 23C
and indefinitely when frozen. The t-PA formulated in this manner was shown to actively mediate the dissolution of blood clots when administered to rabbits and dogs.
Cation exchange chromatography can also be used to concentrate the t-PA. The t-PA eluted from the lysine ~olumn at pH 4 can be directly passed through a column of S-Sepharose-FFtm ~ ¦ra~ <

1 ~Pharmcia, Inc. ~ eguilibrated at ~ with the came ly6ine column ~lution buffer. The t-PA i6 then elutea at pH S.0 (3 ~M Na-glutamate or 2.5 ~M Na citrate, 0.01% Pluronic F-68 containing 200-500 mM NaCl.) Non-ion~c detergenta are ordinarily u6ed during cell extractions and chromatogr~phy to increase t-~A yields ~nd reduce non-specific adsorption; The use of non-ionic detergent 6uch a6 Iween 80 or Triton X-100 to enhance the recovery of t-PA $s well known (Ri~ken et al., ~upra). However, since ~ost common non-ionic detergent6 have critical micellar concentration6 on the order of 0.001%, they can not be effectively removed by cimple dialy6i6, and therefore impede the concentration of aolutions.
Zwitterqent 3-12 work6 effectively in ensuring high yielda of t-PA, ~nd can be used at a concentration of 0.05%, le~s than one-half of it~ critical micellar concentration. Because of lt6relatively high critical micellar concentration, the detergent can be removed effectively by dialy6is or gel filtration. The t-PA can thereby be formulated at the desired concentration with ppropriate ~urfactants (for example Pluronic F-68) added back, if desired, at concentrations appropriate for intravenoua u6e.
We have chosen to use Pluronic F-68 in our final t-PA
formulations. It i6 a more effective detergent at pH 4-5, the optimal range for concentrating t-PA, than i~ Zwittergent 3-12.
Pluronic F-68 al~o ba~ ~ignific~nt advantages over Tween 80 which ia widely u6ed to atabilize t-PA (Ri~ken ~ Collen, aupra). lt ia *Trade mark - 2~ -1 less toxic than Tween 80 and can be lyophilized to a powder, therefore, making it more compatable in pharmaceutical formulations.
Com~o~.~s 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 thrombolytic activity as well. Preparations of t-PA produced by the methods lo 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.
$he 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, c 1 coronary or pulmonary embolism, ~troke and decreased peripheral blood flow.
These compounds can be administered to mammals for veterinary use 6uch a6 with domestic animals, and clinical use in humans in a manner ~imilar to other therapeutic agent6, 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 in~ection. In such cases it will be necessary to have t-PA available in solutions of appropriate concentrations (1 lo to 10 mg/ml or greater). Physiologically acceptable carrier~ or methods for maint~ ng 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 2~ other physiologically acceptable active or inactive materials, or with physiologically suitable carriers such as, for example, - water or normal 6aline. At the concentrations necessary for therapeutic admini6tration it may be necessary to maintain t-PA
with an appropriate detergent in the compound to prevent aggregation of the protein. The compounds can be administered ~ 333 1 63 1 parenterally, for example, by in~ection. In~ection can be subcutaneou6, ~ntravenous, or by intramuscular in~ection. These compounds are desirably administered in pharmaceutically effective amounts and often~as pharmacologically acceptable 6alts ~uch as acid addition 6alts. Such 6alt6 can include, e.g., hydrochloride, hydrobromide, phosphate, 6ulphate, acetate, benzoate, malate, among others.
Com~oul.ds of the present invention can also be used for preparing anti6era for u6e in immunoa6says employing labelled lo reagent6, usually antibodies. These compounds and immunologic reagent6 may be labelled with a variety of labels ~uch as chromophores, fluorophores 6uch as, fluorescein or rhodamine, or radioi60topes 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 6pecifically 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.

1 In addition, t~e t-PA as prepared in this invention when suitably labelled with radioi~otopes Isuch as 131I, 123I, lllIn or 99mTc may prove useful for the detection and localization of thrombi in patients-The following examples are provided by way of illustration, rather than implying any limitation of the sub;ect 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 (Ri~ken and Collen, supra;
Kluft et al, supra), or alternatively, other plasminogen 15 activator producing cells. This conditioned liquid medium was clarified by centrifugation at 10,000 x g for 30 minutes at 4C
or by filtration through low-protein binding membranes (e.g., Gelman Acrodisc 50A) or filter cartridges (e.g., Sartorius, type CA or PH). With the cartridge filters it is considered desirable 20 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 4C, and passed through a chelating 25 Sepharose column complexed with Zn+~ as recommended by the *Trade mark 1 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 1 for a 10 cm bed of Sepharose CL-6B or 300 cm h 1 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 1 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 lo 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 RIU aprotinin per ml. The t-PA activity associated with the degraded forms of the enzyme was eluted with 20 mM Tris-HCl tpH 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 mM
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 ~hown in Figure lA. 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-HCl (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.

1 A column was c~ocen 6uch that approximately 1 liter of resin was available for each 0.2 g of t-PA.
The lysine-Sepharose was wa~hed at 4C with one column volume of 20 mN Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 80, 10 RIU aprotinin per ml at a rate of about 25 cm per hour. The column wa~ then washed with 20 ~M Tris-HCl (pH 7.5), 500 ~M NaCl, 0.05% Zwittergent 3-12, 10 KIU aprotinin per ml until the absor~ance of the eluent buffer was egual to the applied buffer.
Bound plasminogen activator was eluted by washing the column with 20 ~M Tris-HCl, 500 mM NaCl, 50 mM L-ly~ine, 0.05%
Zwittergent 3-12. Approximately two volume equivalents of elution buffer were required to complete the recovery (Figure lB).
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 4C
with 10 mM Tris pH 8.0, S00 mM NaCl, 0.01% Pluronic F-68 until the adsorbance 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 mN NaCl, 0.01% Pluronic F-68.
Bound plasminogen activator was eluted with 3 mM glutamic acid pH 4.0 160 mN NaCl, 0.01~ Pluronic F-68. The t-PA

; ~
c 1 concentration wa6 0.2 - 0.3 mg/ml and the p~ of the eluent was 4.4 + 0.1 (Figure lC).
Thi6 eluted t-PA was concentrated up to 1 mg/ml over an Amicon YM_30tm membrane by pre~sure 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 ~annitol was added per ml. This ~olution was lyophilized and reconstituted by the addition of water without any loss of activity.
Summaries of the purification a t-~A from 6erum-supplemented and serum-free media Are presented in Tables 1 and 2, respectively. The recovered t-PA, when analyzed by gel electrophoresi~ under non-reducing conditions (~aemmli, 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 tir-sue plasminogen activator. Amino acid sequencing ~Applied Biosystems Model 470A Seguencer) indicated the presence of two molecular forms with N-terminal ~equences ~hown in Table 3. The ~equences and the N-terminal heterogeneity i 1 are as reported in the literature. (Wallen et al Eur. J. Biochem 132:681-686 (1983); Pohl et al. Biochemi6try 23:3701-3707 (1984)).
When the procedures described above are used, 5-25% of the total t-PA i8 in the form of degraded t-PA, ~nd therefore recovered in the "Zn AH eluate. If liquid medium containing 6erum, which has not been pre-adsorbed with lysine-Sepharose was u6e, 25 to 100% of tot~l enzyme eluted from the Zn-chelate column is found in the Zn A eluate. Zymographic analysi6 (Granelli-Piperno & Reich, ~upra) of typical "Zn A" and "Zn B" pools are6hown in Figure 3 and demonstrated the separation of low molecular weight forms of t-PA from the bulk of the intact t-PA.

~`` ;

- 3~ -1 Table 1: Recovery of t-PA from 6erum-supplemented medium conditioned by Bowes melamoma cells.
The lysine-Sepharose column was eluted at pH 8.0 a8 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 ~erum 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

st~n~A~d.

Table 2: Recovery of t-PA from serum-free medium 1 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 1 333 ~ 63 1 Table 3: Amino-terminal ~equence 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 pregence or a~sence of three residues. (*) indicates the reported alternative amino terminus.
Cycle No. Major Peak Minor PeakPredicted Sequence 1 Gly Ser Gly lo 2 Ala Tyr Ala 3 Arg Gln Arg 4 Ser Val *Ser S Tyr Ile Tyr 6 Gln Cys Gln 7 Val Arg Val 8 Ile Asp Ile 9 - Glu Cys Arg Lys Arg 11 Asp Thr Asp 12 Glu Gln Glu 13 Lys Met ~ys 14 Thr Ile Thr Gln Tyr Gln 16 Met Gln Met 17 Ile _ Ile 18 Tyr His Tyr 19 Gln Gln Gln - Ser Gln His Gln Ser Example II: Removal of Degraded t-PA
from Recovered t-PA

t-PA was recovered from conditioned media which had been ~upplemented with O.S% serum. The conditioned medium was applied to a Zn~-chelate Sepharose and the t-PA recovered using the ( ~ 3331 63 1 protocol taught in the literature ~Riiken et al, 6upra, Rijken & Collen, ~upra), The recovered t-PA, cont~in~n~ intact and degraded enzyme, wa~ then chromatographed on ly~ine-Sepharose as described above. miS 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 b~n~;~g of t-PA to Zn-chelating Sepharose resin. This material (Figure 4, ~Load") lo was applied to a column of the resin, and washed and eluted as described in Example I. As expected the ma~ority 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.~), 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 - 3g -1 cell enzyme.

- Example IV: Effect of pH on the solubility of t-P~
Aliguots of a ~olution of intact t-PA at a concentration of approximately 0.1 mg/ml were dialyzed to equilibrium again6t 10 mM buffers of several pH values containing 160 mM NaCl and 0.1%
Tween 80. Each ~ample 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 ~hown to contain the same amount of t-PA; however, in those sample6 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
pH (I.U./ml) 4.0 46,000 5.0 46,000 6.0 34,000 7.8 8,500 :
f `
C~

-- 41~ --1 9.3 15,500 10.5 34,000 This experiment demonstrates that at neutral pH values t-PA
ayy~eyates even in relativeiy dilute solutions. Therefore, to concentrate t-P~ for u~e in a phar~aceutical formulAtion weakly buffered solutions of acidic pH ~hould be employed.

Example V: The use of cation eyçhA~ge chromatography for the concentration of t-PA.
The experiments described in Example IV demonstrate that t-PA is maximally ~olu~le 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-Sepharo6e 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 Flowtm was eguilibrated with 0.01 M sodium acetate, lSO 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-l.
The ~o~u~n was washed at 12 ml h 1 with 2.5 ~M sodium citrate, ~00 mM NaCl, 0.1% Pluronic F-68, pH 5.0 until the 1 adsorbancy at 280 nm of the effluent egualed that of the solution applied to the column. The column was eluted at 12 ml hrh 1 with 2.5 mM 60dium citrate, 1 M NaCl, 0.07% Pluronic F-68 at pH 5Ø
~ractions, 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 ~s 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. This t-PA solution was dialyzed to eguilbrium without loss of activity against a solution containing 3 mM glutamic acid, 160 mM NaCl and 0.01~ Pluronic F-68 (pH 4.0). This ~olution is suitable for further concentration by ultrafiltration or direct formulation in a pharmaceutical preparation.

1 Example Vl: Comparison of Adsorbant Substrates Chromatographic resins were 6ynthesized by dissolving approximately one millimole of each of several diaminocarboxylic acids in one ml of 0.1 M sodium bicarbonate. Each acid was added to 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 4C.
The coupling reactions were terminated by the addition of 200 ml of triethanolamine. After an additional 30 minutes of agitation at 4C, 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 6amples 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-aminocaprioic aci*, 0.1% Tween 80. The enzyme activity rec~vered thereby was measured on plasminogen enriched fibrin plates (Haverkatet & Brakman, supra) to calculate the fraction of enzyme bound by the adsorbant. The results, as 6hown in Table 6 below, demonstrated that L-lysine provides the best chromatographic ligand and that a six carbon spacer between the ~olid support and the ligand improved the efficency of t-PA binding.

*Trade mark 1 ~33~ ~

1 Table 6: 8;~ng of t-PA to Immobilized D~aminocarboxylic Acids t-~A Units Bound Immobilized Ligand C~-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. (Riiken & 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-free F-12/DMEM. Serum-free medium, medium supplemented with 0.5%
heat-inactivated fetal bovine 6erum which had been pre-treated with lysine-Sepharose, or medium supplemented with 0.5% heat-i (: `;

1 333 ~ 63 - ~4 -1 activated fetal bo~ine cerum wa~ added to the cells. Aprotinin was added to each of the media 80 that lndividual well~ in the tissue culture plates cont~ne~ 0, 1, 5, 10, 50 or 100 RIU of aprotinin/ml. The plates wére incubated at 37 C for 48 hours.
The media were harvested, clarified by centrifugation and a~sayed for t-PA activities. The total t-PA in each ~ample was determined from the diameter of the zone of clearing effected by 5 ul ~ample placed into a well formed in a plasminogen enriched fibrin plate tHaverkatet and Brakman, ~upra). Neither the choice of medium nor concentration of aprotinin had any effect on total t-PA 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 (lOt 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 ~amples, which contained 10 mM dithiothreitol, were boiled for 7 minutes; then loaded onto a 8.75% polyacrylamide gel. After running the dye front to the bottom, the proteins were electroblotted onto nitrocellulose.
The nitrocellulose was incubated in S% BSA in 10 mM Tris-HCl pH 7.5, 0.9% NaCl for 30 minutes at room temperature, ~nd then incubated with rabbit anti-t-PA (antiserum to denatured human t-PA) (10 microliters ~erum in 10 ml of 10 mM Tris-HCl pH 7.5, 0.9%

~ :`

1 The nitrocellulose was ~n~h~ted in 5% BSA in 10 mM Tris-HCl pH 7.5, 0.9~ ~a~l for 30 minutes at room temperature, and then incubated with ra~bit anti-t-PA (anti6erum to denatured human t-PA) (10 microliter6 6erum in 10 ml of 10 mM Tris-HCl pH 7.5,~0.9%
NaCl 3% ~SA, 0.05% Tween 20) overnight at 4C. The b;n~;n~ of the rab~it ~nti-t-PA was detected using the Yectastain ABCtm (avidin-biotin-horseradish peroxidase complex) kit and 4-chloro-1-naptithol as the substrate for the peroxidase (Figure 5). On the blots, one-chain t-PA i6 seen a6 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 6hown to be the result of the large amount of albumin in the 6ample 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 6erum 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 KIU/ml aprotinin for either ~erum-free medium (Figure 5) or medium containing 0.5% "scrubbed serum"
(Figure 5C). When "non-scrubbed" serum was used even 100 XIU
aprotinin per ml was not adeguate to completely inhibit formation , - ~6 -1 In media cont~ n~ serum the conversion of one-chain t-PA
to two-chain t-PA is a result of proteolytic activities ~nvolving t-PA as ~ sub~trate. 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.
lo A striking difference between tissue plasminogen activators and uro~ 6e is that the former adsorb to fibrin (Thorsen et al supra), which results in a marked enhancement of the activation of plasminogen (Wallen, Prog. Chem. Fibrinolysis Thrombolysis 3:167-181 (1978)). Fragments generated from a CNBr cleavage of fibrinogen (Niewenhuizen et al, Biochim. Biophys. Acta. 755:531-533 (1983)) also timulate 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-22Sl (Kabi), and with or without fibrinogen fragments (Niewenhuizen et al, 6upra). In this 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 ~A405nm), upon hydrolysis by plasmin. The mixture (0.2 units t-PA, 0.2 mM S-2251, 20 ~g/ml ( :

1 CNBr-fragment6 of human fibrinogen) was incubated at 37 and the absorbance change at 40S nm was read at 15 minute intervals.
Activity i6 determined from a plot of adsorbace V6 (time)2 which is linear (Drapier et al, Biochimie 61:463-571 (1979)~.
As is 6hown is Table 7 equivalent amounts of t-PA, ~s defined by egual zones of clearing on a fibrin plate, exhibited very different activities in the S-2251 assay. In the absence of ~
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 enzyme and to a lesser extent that of the two-chain form ~uch 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 i~ the one-chain enzyme a6 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( A405/min2 x 105)Fold Stimulation -FF ~FF

One-cha~n 0.0614.8 250 Two-chain 0.3416.3 48 Degraded 0.08l.S 19 (50,000 mw) f 1 Although the foregoing invention has been described in some detail ~y way of illustration and example for purpo~es of clarity of underst~~g, 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 subseguent formulation in pharmaceutical compositions requires following the essential steps described in the foregoing invention.

Claims (11)

1. A method for recovering intact tissue plasminogen activator (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;
c) 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 immoblized 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.

2. A method of producing a protein in cultured cells grown in a medium containing serum or fractionated serum, comprising pretreating the serum or fractionated serum by contacting the serum or fractionated serum with an insoluble matrix containing pendant lysine groups, whereby substances selected from the group comprising proteolytic substances and other substances having affinity for lysine are removed from the serum or fractionated serum.

3. A method of claim 2, wherein the protein is recombinant (or exogenous to the cell).

4. A method of claim 2, wherein the protein has an affinity for lysine.

5. A method of claim 2, wherein the serum or fractionated serum is diluted prior to pretreatment.

6. A method of claim 5, wherein the serum or fractionated serum is diluted with at least three volumes of water.

7. A method of pretreating serum or fractionated serum for a medium in which cultured cells can be grown for the production of a protein, comprising contacting the serum or fractionated serum with an insoluble matrix containing pendant lysine groups, whereby substances selected from the group comprising proteolytic substances and other substances having affinity for lysine are removed from the serum or fractionated serum.

8. A method of claim 7, wherein the protein is recombinant (or exogenous to the cell).

9. A method of claim 7, wherein the protein has an affinity for lysine.

10. A method of claim 7, wherein the serum or fractionated serum is diluted prior to pretreatment.

11. A method of claim 10, wherein the serum or fractionated serum is diluted with at least three volumes of water.