EP0238551A1 - Procedes de recuperation d'un activateur de plasminogene des tissus - Google Patents

Procedes de recuperation d'un activateur de plasminogene des tissus

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Publication number
EP0238551A1
EP0238551A1 EP86905624A EP86905624A EP0238551A1 EP 0238551 A1 EP0238551 A1 EP 0238551A1 EP 86905624 A EP86905624 A EP 86905624A EP 86905624 A EP86905624 A EP 86905624A EP 0238551 A1 EP0238551 A1 EP 0238551A1
Authority
EP
European Patent Office
Prior art keywords
intact
recited
serum
substrate
degraded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86905624A
Other languages
German (de)
English (en)
Other versions
EP0238551A4 (fr
Inventor
Craig W. Rice
Michael John Morser
Peter Donner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Berlex Laboratories Inc
Original Assignee
Berlex Laboratories Inc
Codon
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Berlex Laboratories Inc, Codon filed Critical Berlex Laboratories Inc
Publication of EP0238551A1 publication Critical patent/EP0238551A1/fr
Publication of EP0238551A4 publication Critical patent/EP0238551A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)

Definitions

  • 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.
  • t-PA tissue plasminogen activator
  • Plasminogen activators have received attention for their role in the fibrinolytic system. These enzymes catalyze the conversion of the proenzyme plasminogen into the proteolyti ⁇ 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 eukaryoti ⁇ 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.
  • tr-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.
  • 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. 5 ⁇ :407-412 (1984)).
  • the published methods do not provide procedures to concentrate t-PA to give useful therapeutic formulations.
  • 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.
  • 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.
  • 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.
  • t-PA tissue plasminogen activators
  • 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.
  • additional adsorbant substrates e.g. adsorbant substrates comprising at least one aminocarboxylic acid
  • 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, Pis ⁇ ataway, N.J.) chromatography.
  • an additional substrate such as, e.g., lysine-Sepha- rose (Pharmacia Fine Chemicals, Pis ⁇ ataway, 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.
  • aprotonin an inhibitor of t-PA proteases
  • Zn chelate has previously been employed for recovering t-PA (Rij en et al. , supra.)
  • 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.
  • 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 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) ) .
  • 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.
  • Zwittergent 3- 12 (Calbiochem, La Jolla, California)
  • Pluroni ⁇ m F-68 BASF
  • Either of these detergents have the desireable property that they can be freeze-dried to a powder along with the t-PA.
  • 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 * ⁇ rt
  • 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-
  • FIG. 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.
  • FIG. 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 - ⁇ n polyacrylamide gel using the Hoeffer "Mighty Small " electrophoresis unit. Electrophoresis was carried out at a constant 150 V.
  • 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.
  • 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.
  • liquid media useful in the present invention would 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.
  • ligands examples include an adsorbant substrate comprising the general formula:
  • 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.
  • ligands such as lysine and propylsulfonate
  • 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.
  • 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.
  • 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.
  • 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.
  • Bowes melanoma cells adsorbed to tissue culture flasks (Rijken and Collen, supra) or microcarriers (K Kunststoff et al. , supra) were used to condition liqui media which contained 0 to 0.5% scrubbed serum.
  • 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% Pluroni ⁇ 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.
  • 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.
  • 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 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;
  • 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.
  • 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.
  • 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) .
  • 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.
  • These plasminogen activators could be specifically inhibited and immunopre ⁇ ipitated 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 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
  • 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
  • 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.
  • 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.
  • 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) .
  • an aminocarboxylic acid e.g., lysine
  • spacer e.g. a six carbon aliphatic spacer
  • support substrate e.g. Sepharose
  • certain benefits of the present invention can be obtained with any compound wherein both an amino and ⁇ arboxyl 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- hexan ⁇ ic 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.
  • 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.
  • 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.
  • 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-hexanoi ⁇ acid, 20-50 mM L-lysine or 100-300 mM L-arginine.
  • 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
  • tissue plasminogen activator by means of a competitive agent
  • 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.
  • 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 Ami ⁇ on 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.
  • mannitol 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.
  • 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) .
  • 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.
  • 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.
  • appropriate surfactants for example Pluronic F-68
  • 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.
  • t-PA thrombolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity fibrinolytic activity
  • fibrinolytic activity and, therefore display thrombolyti ⁇ 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
  • 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.
  • t-PA 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.
  • 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.
  • 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.
  • labelled reagents usually antibodies.
  • These compounds and immunologi ⁇ 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.
  • 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.
  • 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) .
  • 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; K Kunststoff 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 ⁇ r PH) .
  • low-protein binding membranes e.g., Gel an Acrodisc 50A
  • filter cartridges e.g., Sartorius, type CA ⁇ r PH
  • 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.
  • 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) .
  • 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 ads ⁇ rbance 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.
  • 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.
  • 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)) .
  • Lysine- Sepharose 28 1.3 739,000 75
  • 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.
  • 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.
  • 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 -
  • 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.
  • each sample 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.
  • Example V The use of cation exchange chromatography for the concentration of t-PA.
  • 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.
  • cation exchange resins such as SP-Sepharose or S-Sepharose-FF.
  • 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) .
  • Chromatographi ⁇ 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 t ⁇ 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.
  • 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% polya ⁇ rylamide 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%
  • rabbit anti-t-PA anti-denatured human t- PA
  • 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) .
  • Vectastain ABC avidin-biotin-horseradish peroxidase complex
  • 4-chloro-l naptithol as the substrate for the peroxidase
  • Example VIII The stimulation of "intact"
  • tissue plasminogen activators and urokinase are tissue plasminogen activators and urokinase.
  • 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 also stimulate the process of plasminogen activation by t-PA.
  • 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) .
  • 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 (A 405 ) , upon hydrolysis by plasmin.
  • the mixture (0.2 units t-PA, 0.2 mM S-2251, 20 ⁇ g/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)).

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Abstract

Un procédé de récupération d'un activateur de plasminogène des tissus t-PA à partir d'un milieu liquide est décrit. Le procédé consiste à mettre en contact un milieu liquide avec au moins un substrat capable d'effectuer une séparation de t-PA intacte de t-PA dégradée puis à récupérer le t-PA intact libre de toute autre protéine sans lien avec l'activateur. La présente invention divulgue également des composés produits à l'aide de ce procédé, des composés comprenant un t-PA intact à une chaîne et des compositions pharmaceutiques les contenant ainsi que des procédés utilisant de telles compositions.
EP19860905624 1985-09-06 1986-09-05 Procedes de recuperation d'un activateur de plasminogene des tissus. Withdrawn EP0238551A4 (fr)

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DE3512910A1 (de) * 1985-04-11 1986-10-16 Behringwerke Ag, 3550 Marburg Verfahren zur reinigung von plasminogenaktivatoren
US4960702A (en) * 1985-09-06 1990-10-02 Codon Methods for recovery of tissue plasminogen activator
US4898825A (en) * 1986-05-07 1990-02-06 Mitsui Toatsu Chemicals, Incorporated Methods for purification of single-chain and double-chain tissue plasminogen activator
PT84991B (pt) * 1986-06-06 1990-03-08 Genentech Inc Processo para a producao de actividador do plasminogenio biologicamente activo
WO1988000615A1 (fr) * 1986-07-16 1988-01-28 Celltech Limited Procede de purification d'un activateur de plasminogene
JPS6463378A (en) * 1986-08-11 1989-03-09 Mitsui Toatsu Chemicals Separation of single stranded tpa and double standard tpa
JPS6379591A (ja) * 1986-09-22 1988-04-09 Mitsui Toatsu Chem Inc ｔＰＡの精製方法
AU1668988A (en) * 1987-06-03 1988-12-08 Smithkline Beckman Corporation Purification of tpa
US4929560A (en) * 1988-02-03 1990-05-29 Damon Biotech, Inc. Recovery of tissue plasminogen activator
WO1990002175A1 (fr) * 1988-08-16 1990-03-08 Novo Nordisk A/S Procede de production de polypeptides par culture de cellules eukaryotiques en presence d'inhibiteurs de protease
US4980165A (en) * 1989-01-27 1990-12-25 Genetics Institute, Inc. Pharmaceutical formulations of plasminogen activator proteins
DK0485504T3 (da) * 1989-08-11 1994-04-18 Zymogenetics Inc Fremgangsmåde til fremstilling af aktiveret protein C ved celledyrkning

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US4245051A (en) * 1978-03-30 1981-01-13 Rockefeller University Human serum plasminogen activator
NL8003402A (nl) * 1980-06-11 1982-01-04 Leuven Res & Dev Vzw Nieuwe plasminogeen-activator en farmaceutisch preparaat met trombolytische werking.
DE3584902D1 (de) * 1984-02-29 1992-01-30 Asahi Chemical Ind Waessrige loesung eines darin in erhoehter konzentration aufgeloesten gewebe-plasminogen-aktivators und herstellungsverfahren.
AU593264B2 (en) * 1985-07-10 1990-02-08 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Chromosomal DNA sequence, expression vector for human tissue plasminogen activating factor, cultured cells transfected with same and method of producing said activating factor

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