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WO1990001333A1 - PROCEDE DE PREPARATION DE COMPOSITIONS DE tPA - Google Patents

PROCEDE DE PREPARATION DE COMPOSITIONS DE tPA Download PDF

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Publication number
WO1990001333A1
WO1990001333A1 PCT/US1989/003301 US8903301W WO9001333A1 WO 1990001333 A1 WO1990001333 A1 WO 1990001333A1 US 8903301 W US8903301 W US 8903301W WO 9001333 A1 WO9001333 A1 WO 9001333A1
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Prior art keywords
tpa
type
composition
support matrix
activity
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Inventor
William R. Tolbert
Christopher P. Prior
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Invitron Corp
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Invitron Corp
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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 methods for pre ⁇ paring pharmaceutical compositions of active polypep- tides. More specifically, this invention relates to a method for preparing compositions of tPA and an excipient protein by co-purification.
  • tPA Human tissue plasminogen activator
  • tPA Human tissue plasminogen activator
  • Plasmin degrades the fibrin matrix of blood clots, thereby restoring the hemodynamic condition of an open vascular system after an internal vascular accident has produced thrombosis or thromboembolism.
  • Vascular dis ⁇ ease states involving partial or total blockage of blood vessels which are amenable to treatment with plasminogen activators include, stroke, pulmonary embolism, myocar- dial infarction, as well as deep vein and peripheral artery obstructions.
  • tPA is one of two immunologically distinct types of plasminogen activators found in human plasma and other body fluids.
  • tPA is considered to be the physiologically relevant thrombolytic agent and is considered to have significant potential as a therapeu ⁇ tic agent.
  • tPA thrombolytic agent Due to the extremely low concentration of tPA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought.
  • Preferred methods of production for commercial utilization involve growing cells capable of producing tPA in culture, including cells transformed with re- combinant vectors which encode this protein.
  • a pre ⁇ ferred method for producing tPA including that ex ⁇ pressed from recombinant DNA vectors, is in mammalian cell culture systems. Mammalian cells have the capabilities ⁇ ity to synthesize human proteins with the proper con ⁇ figuration, correct disulfide bonding, and arrays of carbohydrate side chains which all result in a protein which mimics the naturally occurring protein in terms of activity and lack of immunogenicity.
  • tPA has been syn ⁇ thesized in a variety of cells, including those trans ⁇ formed with recombinant expression vectors containing sequences which encode tPA and its derivative analogs. See, for example, Van Brunt, Biotechnology (1986) :505.
  • the procedures for cell culture utilizing the different ap ⁇ paratus may be classified as batch procedures, semi- batch procedures, and continuous or perfusion proce ⁇ dures.
  • batch procedures the cells are grown in a fixed amount of nutrient and harvested after significant growth ceases; the desired product is isolated from the cell culture after the first round of growth.
  • Semi- batch procedures are similar to batch procedures; how ⁇ ever, after harvest the cells are submitted to repeated rounds of growth and expression by resuspension in fresh medium , .
  • 4,752,603 dis ⁇ closes a process for preparing a tPA-like protein from human melanoma cell cultures, and a process for its pu ⁇ rification.
  • Collen disclosed purification using a zinc chelate agarose ligand exchange column, followed by affinity chromatography on lectin agarose, optionally followed by gel filtration, preferably using cross- linked dextran particles.
  • Collen also disclosed addi ⁇ tion of aprotinin to the various solutions during puri ⁇ fication (except for the last step) to inhibit proteo- lysis of the tPA during purification.
  • Collen reported a yield of 46% of tPA having a specific activity of about 500,000 IU/ g, based on WHO First International Refer ⁇ ence Preparation of tPA.
  • Michio et al, EPO 217,379 disclosed formu ⁇ lations designed to increase the low aqueous solubility of tPA- These formulations include large quantities of arginine, optionally NaCl, and may include additional, proteins as excipients.
  • Michio disclosed an exemplary formulation containing 5x10 IU tPA, 52.5 mg arginine HC1, and 20 mg HSA.
  • tPA can be extracted from a variety of tissues and has been purified from pig heart (P. Wallen et al, Biochim Biophys Acta (1982) 1 :318-328). A recent review of tPA and its characterization is found in A. Klausner, Biotechnology (1986) :706-71.
  • This assay involves the catalyzed conversion of plasmin ⁇ ogen to plasmin and measurement of the plasmin formed colorimetrically.
  • the activity of all tPA preparations in this assay can be enhanced by addition of cyanogen bromide fragments of fibrinogen, by the addition of fi- brinogen itself, or by the addition of fibrin.
  • Other useful in vitro assay systems include the direct meas ⁇ urement of lysis of fibrin clots as described by P. Wallen et al, Biochim Biophys Acta (1982) (supra) . In vivo assays are also available.
  • tPA isolated from Bowes melanoma cells is separable into two "types", Type I. and Type II, by fractionation with arginine- or lysine-conjugated Sepharoses. These types of tPA differ in glycosylation pattern; Type I tPA is glycosylated at each of three canonical asparagine glycosylation sites; Type II has glycosylation at only two of these sites.
  • carbohydrate composition therefore resides in the A chain; when tPA is cleaved, typically in the presence of plasmin, cleavage occurs at the proteolytic site between arg 275 -ile_ 76 (G. Pohl et al, EMBO Workshop on Plasminogen Activators, Amalfi, Italy, (October 1985)). In addition, P.
  • Type I and Type II tPA have been reported as partially separated on arginine-Sepharoses or lysine Sepharoses.
  • tPA is co-purified with an excipient protein such as human serum albumin (HSA), to provide directly a composition of suitable concentration for administration to mammals, including humans.
  • HSA human serum albumin
  • Control sequence refers to DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending on the host organ ⁇ ism; in eukaryotes, generally, such control sequences include promoters, terminators, and in some instances, enhancers.
  • control sequences is intended to include . , at a minimum, all components whose presence is necessary for expression, and may also include addi ⁇ tional components whose presence is advantageous to the expression of the desired gene.
  • “Operably linked” refers £0 a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • “Expression system” refers to a DNA segment which contains at least one "expression cassette", which cassette contains a coding sequence to be expressed and the control sequences, operably linked to it, which enable its expression.
  • Effective refers to cell culture medium con ⁇ taining tPA synthesized by cells contained in a cell culture vessel.
  • the terms “effluent” and “conditioned medium” are used interchangeably herein.
  • support matrix refers to a gen ⁇ erally inert medium which can be derivatized as desired.
  • Support matrices within the scope of this invention include, without limitation, agarose, Sepharoses, Sephadexs>, Sephacol?, polyacrylamide, cellulose (and cellulose derivatives), and membrane-type supports, such as nylon, polystyrene, and the like, including hollow fibers .
  • negatively-charged ionic group refers to a radical which bears a negative formal charge under the pH conditions used herein, and which does not react irreversibly with the tPA or HSA to be eluted.
  • Preferred negatively-charged ionic groups are easily attached to support matrices, or are commercially avail ⁇ able in such form.
  • Exemplary negatively-charged ionic groups include without limitation sulfonic, phospho, carboxy, carboxymethyl, and the like. Sulfonic groups are presently preferred.
  • basic amino acid refers to an a ino acid having a side chain which accepts protons at the pHs used herein.
  • exemplary basic amino acids include lysine and arginine, preferably lysine. Histidine may also be employed.
  • mammalian serum albumin refers to the serum albumin from any convenient mammal, for example human, bovine, ovine, equine, murine, etc.
  • Human serum albumin (HSA) is presently preferred, par ⁇ ticularly for compositions intended for administration to humans.
  • serum albumin is purified away from tPA in the process of preparing purified Type I and Type II tPA, serum albumin from other species may be used in these processes.
  • serum-free media and “substan ⁇ tially serum-free” refer to cell culture media which contain serum in amounts less than about 1%, preferably less than about 0.1%, and most preferably contain no serum at all. Serum-free media sufficient for basal cell culture are commercially available.
  • the medium is preferably supplemented with insulin and transferrin (in addition to MSA, preferably HSA) , and additionally contains an antibiotic such as methotrexate.
  • tPA refers to the 527 amino acid protein having the primary structure disclosed by Pennica et al, (supra) and its naturally occurring variants, including allelic variations.
  • Type I tPA is defined as the foregoing primary structure which con ⁇ tains glycosylation at three sites which correspond to positions 117, 184 and 448 in the sequence described by Pennica.
  • Type II tPA is defined as tPA containing glycosylation only at sites corresponding to positions 117 and 448 of the Pennica structure.
  • a "substantially pure mixture" of HSA and tPA refers to a composition which contains essentially only tPA (as Type I and/or Type II) and human serum albumin. Other proteins (e.g., from the cell culture medium) may be present in small amounts, not exceeding about 3% for any given protein. At least 95% of the composition will be HSA and tPA.
  • the substantially pure mixture will also be acceptable for administration to humans by parenteral means.
  • the mixture has a specific activity of about 50,000-400,000 IU/mg protein, preferably 100,000-300,000 IU/mg protein.
  • high yield means a yield of pharmaceutically acceptable tPA in excess of 50%, preferably greater than 60%, based on the amount of tPA present in the crude conditioned media. Previous reports of tPA purification fail to recover even 50% of the tPA initially present in conditioned media; yields in the range of 35-46% are the maximum reported. The purification process of the instant invention provides purified tPA in excess of 50% yield.
  • Type I and Type II tPA occur in extracts from a variety of mammalian tissue sources. While it is known that the glycosylation of the molecule varies in the structure of the carbohydrate moieties attached at these positions depending on the cells producing the protein, it is nevertheless the case that the distribution of glycosylation with respect to these canonical sites is the same regardless of source. Thus, tPA recombinantly produced in mammalian cells, such as Chinese hamster ovary (CHO) cells or mouse cells; native tPA obtained from colon cells, uterine cells, or melanoma cells, etc. exhibits both Type I and Type II variants. The glycosylation differences among tPA prepared from these various sources reside in the structures of the carbohydrate chains themselves.
  • Specific activity in IU/mg is defined herein by comparison to the International Standard tPA obtained from the National Institute of Biological Standards, England.
  • the International tPA standard is a lyo- philized preparation of tPA Type I and Type II, unsepar- ated, having a total of 1000 IU per vial.
  • the absolute activity of tPA is highly dependent on the conditions of the assay, for example in the in vitro assay measuring conversion of plasminogen to plasmin referenced above, addition of various materials increases the apparent activity. However, the specific activity relative to the standard will not change by virtue of this varia ⁇ tion.
  • the standard tPA preparation exhibits the same variation with regard to conditions, and thus the activity relative to it remains the same unless the inherent character of the protein has been changed.
  • differences in the specific activity of Type I and Type II preparations are normalized with regard to the mixture — i.e., the standard.
  • Immulon II 96-well microtiter plates are used. Into each well are placed 200 ul of 10 ug/ml solution in water of fibrinogen (from which plasminogen had been removed by lysine Sepharoses- column) . The plates are allowed- to air dry overnight. To obtain clotting, 200 ul/well bovine thrombin is added (Sigma) supplied as a 1 NIH unit/ml solution in PBS containing 0.1% BSA and 1 mM CaCl- . The plates are left 45 minutes at room tem ⁇ perature, and then rinsed with saline Tweens, followed by two 30 minute washes with saline Tweens. The final wash is shaken out and the plate is again air dried. The dried plates can be stored refrigerated.
  • test buffer is Tris saline prepared by dissolving 605 mg Tris in 800 ml distilled water and adjusting the pH to 8.8 with 6 N HC1, then dissolving 5.84 g NaCl and 0.1 g Triton X-100 into the buffer and diluting to 1 L.
  • the types of cells used are any cells which are capable of producing tPA at levels suitable for commercial production, i.e., preferably at least about 1 ug tPA/10 cells/day, and which secrete the product into the cell culture medium.
  • a variety of cells which are capable of expressing tPA are known in the art. For example, rat prostate adenocarcinoma cells (U.S. Pat. No. 4,661,453), and a human melanoma cell line (Bowes, EPO 41,766, published Dec. 16, 1981); Rijken and Collen (1981), and K Kunststoff et al (1983)) have been shown to synthesize tPA.
  • the cells expressing tPA may also be cells transformed with recombinant vectors containing a nucleotide sequence encoding tPA operably linked to control sequences which allow its expression and secre ⁇ tion.
  • Transformed cells of this type include bacterial cells, for example, transformed E. coli cells, trans ⁇ formed B. subtilis cells, and the like.
  • fungal cells for example, yeast cells. See, for example, EPO No. 85306432.7.
  • Mammalian cells may also be transformed with a recombinant vector which contains an expression system which encodes tPA.
  • tPA may be by Chinese hamster ovary cells (CHO) transformed with a vector pSTH-MDH, which contains the tPA sequences and an ampli- fiable DHFR gene.
  • pSTH-MDH Chinese hamster ovary cells
  • the construction of pSTH-MDH and the CHO host cell line are described in commonly owned USSN 010,871; examples of other vectors which may also be used to transform mammalian cell ' lines are also de ⁇ scribed in this application, and include pSV-tPA17, and pSTH-SDH.
  • Another vector which may be used to transform CHO cells is that described by R.J. Kaufman et al, J Mol Biol (1982) , 159:601.
  • the cell culture may be grown in any accept ⁇ able system, for example, perfusion, batch, and semi- batch.
  • Perfusion systems are presently preferred.
  • medium is perfused through the reactor at a rate proportional to the number of cells and their metabolic characteristics.
  • Process parameters can be maintained by introducing reagents and gases directly into the vessel or into the medium as it is being fed to the vessel.
  • Effluent is removed from the perfusion chamber on a continuous or semi-continuous basis. Examples of perfusion systems are described in Tolbert et al. (1985), Tolbert et al (1987), and compo ⁇ nent parts in U.S. Pat. Nos.: 4,166,768; 4,178,209; 4,184,916; 4,537,860; and 4,335,215.
  • Mammalian cells which grow in suspension cul ⁇ ture may be used to produce tPA in a perfusion culture system, as described in U.S. Pat. Nos. 4,166,768; 4,178,209; and 4,184,916.
  • a constant environment is maintained in the growth vessel by con ⁇ tinually adding fresh nutrients and removing waste prod ⁇ ucts at a rate proportional to the number of cells in the vessel.
  • the system can operate for prolonged periods of time with cell densi ⁇ ties from 10 to 30 times the maximum cell density in a typical batch reactor.
  • a gentle "sail" agitator system is used to prevent cell damage at these high cell densi ⁇ ties.
  • the filtration unit separates the cells from the product-containing cell conditioned medium and returns the cells to the growth vessel as fresh medium is added. Dissolved oxygen and carbon dioxide, as well as pH levels, are continually monitored and maintained throughout the production run.
  • the effluent from the reactor contains waste products and the desired protein products secreted by the cells. Product reservoirs may be removed periodically for further processing by con ⁇ centration and other conventional protein purification techniques.
  • the suspension culture perfusion system maintains an optimal cell den ⁇ sity in the growth vessel by transferring excess cells to the harvest vessel. After the optimal cell density has been attained in the growth vessel, the serum con ⁇ centration in the fresh medium can be significantly reduced.
  • the cells are cultured in defined media which does not contain serum.
  • serum-free media containing a mam ⁇ malian serum albumin (preferably HSA)
  • the resulting tPA is produced with an enhanced Type II/Type I ratio.
  • serum-free HSA media we have obtained tPA compo ⁇ sitions containing >50% Type II, as compared with the products of serum-containing media which typically pro ⁇ vide tPA that is about 40% Type II.
  • the amount any type of tPA in the effluent may be determined by any method which conveniently distin ⁇ guishes the species, for example, by the difference in molecular weight on gel filtration or upon gel electro- phoresis using Laemmli gels under non-reducing condi ⁇ tions.
  • the presence of tPA in the fractions obtained by molecular weight sizing materials can be detected by procedures known in the art, for example, by determi ⁇ nation -of. the enzymic tPA activity, or by immunoassay- An Elisa assay for tPA is described in P.G. Abrams et al, J Immunol (1984), 132:1611, and kits providing the reagents for Elisa assays of tPA are commercially avail ⁇ able.
  • wild-type mono- meric tPA is synthesized as a single chain proenzyme of approximate molecular weight of 70 kd, which can be cleaved by plasmin or trypsin, without disrupting the disulfide linked two-chain molecular structure.
  • tPA dissociates into a heavy and a light chain, with approximate molecular weights of 35,000.
  • Wild type tPA contains two kringles linked by a hexapep- tide linker sequence.
  • a kringle is a triple disulfide- linked sequence of amino acids which form a loop. These kringles are believed to be responsible for binding of tPA to fibrin (Thorsen, Biochim Biophys Acta (1975), 3_92:55) .
  • the invention provides highly purified forms of Type I and Type II tPA through separation of a puri ⁇ fied tPA/HSA mixture.
  • higher final yields of purified tPA are attained.
  • the conditioned media are cooled from about 37 C (or the cell culture temperature) to about 4 C and the pH lowered to 5.0 with dilute acetic acid.
  • the media is then applied to a cationic exchange resin, having a negatively-charged ionic group bound to a support matrix.
  • Suitable ionic groups include sulfate and carboxymethyl, preferably sulfate.
  • Suitable support matrices include agarose, Sepharoses>, Sephadexs, Sephacol ⁇ ', polyacrylamide, cellulose, membranes, and the like. Presently preferred is Fast-Flow S Sepharoses.
  • the resin is pre-equilibrated with 50 mM NaOAc/100 mM NaCl, pH 5.0 to adsorb the tPA and HSA.
  • the adsorbed tPA/HSA is eluted with 20 mM Tris/1 M NaCl pH 8.0.
  • the resulting material comprises a substantially pure mixture of HSA and tPA, having an activity of about 50,000 to about 400,000 IU/mg protein (preferably about 100,000 to 300,000 IU/mg). This is a suitable concentration for administration to mammals, including humans.
  • the prod ⁇ uct at this point may be purified further to provide pure Type I and Type II tPA.
  • the compo ⁇ sition may be sterile-filtered, dialyzed (optionally lyophilized) , and packaged for administration.
  • anionic ion exchange e.g., Fast-Flow Q Sepharoses *
  • the eluted tPA/HSA is concentrated, for example by pre ⁇ cipitation with salts, ultrafiltration, etc.
  • a clean separation into Type I and Type II tPA is then made using a support matrix conju ⁇ gated to a basic amino acid, preferably a lysine- Sepharosei- column.
  • This process differs from the process dis ⁇ closed in copending USSN 178,392 in that it eliminates the steps of PABA-Sepharoses> adsorption and Sephacryls filtration, and results in enhanced yield of tPA without reduction in specific activity.
  • tPA produced by the processes of the invention may be used for the purposes already described for tPA, including therapy for disorders related to blood clots.
  • the product will be formu ⁇ lated in pharmacologically acceptable compositions, and administered in therapeutically acceptable doses, as described in commonly-owned copending U.S. application 130,901, filed 10 December 1987 and specifically incorporated herein by reference in full.
  • Effective dosages and administration regimens are known in the art.
  • the precise dosage administered will depend upon the species, age, size, and general condition of the subject, as well as the exact nature and severity of the condition to be treated. Thus, a precise effective dose cannot be specified in advance.
  • an ef ⁇ fective dose for treating thrombosis for example, myo- cardial infarction, deep vein thrombosis, etc.
  • This dose is preferably administered by bolus injection i.v., but may alternatively be accomplished by slower i.v. infusion.
  • Polyacrylamide gel electrophoresis is carried out as described by Laemmli (1970) in the presence or absence of sulfhydryl reducing reagents.
  • Immunoblotting techniques and Elisa assays for the' detection and/or quantitation of tPA are performed as described by Abrams supra, using as antibody a mouse IgG. monoclonal antibody which recognizes an epitope in the A chain of human tPA.
  • PCV packed cell volume
  • Example 1 (Production of tPA in Cell Culture)
  • CHO cells transformed with the vector pSTH-MDH, as described in commonly owned USSN 010,871 are grown in any suitable, commercially available basal growth media, preferably a medium which does not contain serum.
  • Suitable media include, without limitation, Ham's F-12, DMEM, IMDM and the like.
  • Mammalian serum albumin, preferably HSA is added to the medium at a con ⁇ centration of about 0.1% to about 0.2%, and further sup ⁇ plemented with methotrexate, insulin, and transferrin.
  • Cells are grown in a 100 L perfusion system as described in commonly-owned copending application. USSN 092,502, filed 3 September 1987, incorporated herein by reference in full.
  • the rate of perfusion is balanced to the PCV in an effort to deliver nutrients and remove waste products at a rate compatible with cell me ⁇ tabolism.
  • the cells are allowed to grow to a PCV of about 10-20 ml/L, to maximize production of tPA.
  • the effluent is immediately transferred to 4°C upon collection.
  • a control group of cells are cultured under the conditions described in part (A) above, but replacing the HSA added to the medium with 5% fetal calf serum.
  • a 5-liter S-Sepharoses- Fast-Flow bioprocess column (Pharmacia; 25.2 cm x 10 cm) is equilibrated with five column volumes of buffer #1 at 150 1/h.
  • Cell cul ⁇ ture conditioned media are pH adjusted to 5.0 with dilute acetic acid. After adjustment, the media are applied to the column at the same flow rate.
  • the pH and conductivity of the media are such that tPA and HSA bind selectively to the column electrostatically while the bulk of the serum proteins and non-proteinaceous material (nucleic acids, phospholipids, etc.) do not bind to the resin and are removed in the column flow- through.
  • the tPA/HSA mixture is eluted fro the column with about 4 column volumes of buffer #2.
  • the resulting composition consists essentially of tPA (as a mixture of Type I and II) and HSA, having an activity of 50-400 x 10 IU/mg (preferably 100,000- 300,000 IU/mg).
  • the composition is in a form suitable for packaging, and may if desired be injected. I.e., this composition is in stable form, and does not require the addition of supplementary proteins to enhance stability and bulk, etc.
  • one may further purify the mixture without substantially altering the specific activity by treatment with an additional ion exchange resin (preferably Fast-Flow Q Sepharoses) and gel fil ⁇ tration.
  • the composition may be suspended in saline or other suitable injection carriers for intravenous administration.
  • the gel filtration step may conveniently be used to change buffers to one acceptable for immediate administration.
  • Example 2(A) The composition prepared in Example 2(A) is concentrated by precipitation with ammonium sulfate (550 g/1) at 4°C for a minimum of 4 hours. The precipitate is collected by centrifugation (5000 rpm for ap ⁇ proximately 30 minutes). The pellets may be stored at - 70°C, if necessary. The pellet is then dissolved in buffer #3, and clarified by centrifugation and/or fil ⁇ tration.
  • ammonium sulfate 550 g/1
  • the precipitate is collected by centrifugation (5000 rpm for ap ⁇ proximately 30 minutes).
  • the pellets may be stored at - 70°C, if necessary.
  • the pellet is then dissolved in buffer #3, and clarified by centrifugation and/or fil ⁇ tration.
  • the purified tPA thus obtained is then sep ⁇ arated cleanly into Type I and Type II tPA using a 2.6 x 100 cm column of lysine Sepharose® to selectively adsorb the tPA.
  • the column is pre-equilibrated with buffer #3 and the tPA loaded onto the column in amounts ranging from about 50-800 mg. Elution is effected by applica ⁇ tion of a gradient of buffer #3 and #4 which results in a clean separation of Type I and Type II tPA in each case.
  • Each of the peaks containing Type I and Type II tPA is assayed according to the procedure set forth above using the International Standard of tPA obtained from the National Institute of Biological Standards as specified above, or a secondary standard compared thereto.
  • the protein eluted as Type I tPA shows an activity of 420 x 10 IU/mg, within experimen ⁇ tal error; that eluted as Type II tPA shows a specific activity of about 550 x 10 IU/mg within experimental error.
  • the yield obtained is superior to the yields reported in the prior art (including copending USSN 178,392). This may be due in part to the simplification of the purification process; however, the fact that intermediate steps in purification may be omitted with ⁇ out adverse impact on the final purity of the product was unexpected.
  • the tPA elaborated in serum-free media containing serum albumin contains a greater ratio of Type II to Type I tPA than the tPA elaborated in media containing FCS.
  • the control (FCS) tPA generally exhibits a ratio of II/I of about 40/60, whereas the process of the invention yields a ratio of about 60/40 or greater.
  • FCS control
  • This improved culture condition should generally provide tPA that is greater than 50% Type II, preferably 60% or greater Type II.
  • Formulations suitable for administering tPA to a subject may be prepared as follows: (A) Approximately 50 mg of purified tPA (Type 1)
  • Example 3 in the elution buffer remaining from Example 3 is transferred via gel filtration to a solution of 150 mM arginine, 50 mM sodium citrate, 20 mg/ml HSA, pH 6.0,
  • the solution may be sterile filtered if desired, and packaged for storage.

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Abstract

On produit avec un bon rendement de l'activateur plasminogène de tissu (tPA) dans des cultures cellulaires par perfusion à l'aide de HSA (albumine de sérum humain) dans des milieux dépourvus de sérum. On peut purifier les milieux conditionnés ainsi obtenus à l'aide de SépharoseR afin de produire une composition pouvant être injectée. On peut purifier davantage la composition afin de produire du (tPA) de type I et de type II à haut rendement. Etonnament, la culture dans des milieux dépourvus de sérum contenant de l'HSA, fournit un produit de tPA enrichi dans le tPA de type II plus actif.
PCT/US1989/003301 1988-08-02 1989-07-31 PROCEDE DE PREPARATION DE COMPOSITIONS DE tPA Ceased WO1990001333A1 (fr)

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US22752988A 1988-08-02 1988-08-02
US227,529 2009-07-22

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WO1990001333A1 true WO1990001333A1 (fr) 1990-02-22

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PCT/US1989/003301 Ceased WO1990001333A1 (fr) 1988-08-02 1989-07-31 PROCEDE DE PREPARATION DE COMPOSITIONS DE tPA

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AU (1) AU4187389A (fr)
WO (1) WO1990001333A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991008766A1 (fr) * 1989-12-20 1991-06-27 Boehringer Mannheim Gmbh STABILISATION DE t-PA pro
WO1991008767A1 (fr) * 1989-12-20 1991-06-27 Boehringer Mannheim Gmbh STABILISATION DE t-PA GLYCOSYLE
EP0458950B1 (fr) * 1989-12-20 1993-09-15 Roche Diagnostics GmbH Stabilisation de k2p pro
US20130058939A1 (en) * 2009-08-31 2013-03-07 Inserm Treatment of neurological or neurodegenerative disorders
CN108424897A (zh) * 2018-03-13 2018-08-21 广州铭康生物工程有限公司 一种rhTNK-tPA细胞收获液的纯化方法

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Publication number Priority date Publication date Assignee Title
US4245051A (en) * 1978-03-30 1981-01-13 Rockefeller University Human serum plasminogen activator
US4550080A (en) * 1984-06-05 1985-10-29 Asahi Kasei Kogyo Kabushiki Kaisha Process for the preparation of a plasminogen activator
EP0261941A2 (fr) * 1986-09-22 1988-03-30 MITSUI TOATSU CHEMICALS, Inc. Procédé de purification d'activateur de plasminogène tissulaire
US4757005A (en) * 1984-04-19 1988-07-12 Miles Inc. Method and cell line for obtaining plasminogen activators
US4766075A (en) * 1982-07-14 1988-08-23 Genentech, Inc. Human tissue plasminogen activator

Patent Citations (5)

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US4245051A (en) * 1978-03-30 1981-01-13 Rockefeller University Human serum plasminogen activator
US4766075A (en) * 1982-07-14 1988-08-23 Genentech, Inc. Human tissue plasminogen activator
US4757005A (en) * 1984-04-19 1988-07-12 Miles Inc. Method and cell line for obtaining plasminogen activators
US4550080A (en) * 1984-06-05 1985-10-29 Asahi Kasei Kogyo Kabushiki Kaisha Process for the preparation of a plasminogen activator
EP0261941A2 (fr) * 1986-09-22 1988-03-30 MITSUI TOATSU CHEMICALS, Inc. Procédé de purification d'activateur de plasminogène tissulaire

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Title
(KRUITHOF), "Human Tissue-Type Plasminogen Activator", BIOCHEM J,. issued 1985, Volume 226, pages 631-636, see abstract and pages 633-634. *
(RANBY), "Isolation of two variants of native one-chain tissue plasminogen activator", FEBS LETTERS, issued September 1982, Volume 146, Number 2, pages 289-292, see the entire document. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991008766A1 (fr) * 1989-12-20 1991-06-27 Boehringer Mannheim Gmbh STABILISATION DE t-PA pro
WO1991008767A1 (fr) * 1989-12-20 1991-06-27 Boehringer Mannheim Gmbh STABILISATION DE t-PA GLYCOSYLE
EP0458950B1 (fr) * 1989-12-20 1993-09-15 Roche Diagnostics GmbH Stabilisation de k2p pro
US20130058939A1 (en) * 2009-08-31 2013-03-07 Inserm Treatment of neurological or neurodegenerative disorders
CN108424897A (zh) * 2018-03-13 2018-08-21 广州铭康生物工程有限公司 一种rhTNK-tPA细胞收获液的纯化方法
CN108424897B (zh) * 2018-03-13 2020-08-14 广州铭康生物工程有限公司 一种rhTNK-tPA细胞收获液的纯化方法

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