WO1989009820A1 - tPA DE TYPE I ET DE TYPE II PURIFIE - Google Patents

tPA DE TYPE I ET DE TYPE II PURIFIE Download PDF

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Publication number
WO1989009820A1
WO1989009820A1 PCT/US1989/001406 US8901406W WO8909820A1 WO 1989009820 A1 WO1989009820 A1 WO 1989009820A1 US 8901406 W US8901406 W US 8901406W WO 8909820 A1 WO8909820 A1 WO 8909820A1
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Prior art keywords
tpa
type
specific activity
cho
derived
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PCT/US1989/001406
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English (en)
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George D. J. Green
Christopher P. Prior
Garance Prior
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Invitron Corporation
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Publication of WO1989009820A1 publication Critical patent/WO1989009820A1/fr

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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
    • 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

  • the invention relates to the recovery of purified forms of tissue plasminogen activator proteins.
  • the invention concerns isolated Type I and Type II tPA having specific activities on the order of 500 ⁇ 10 3 IU/mg.
  • Tissue plasminogen activator is a highly effective, fibrin dependent enzyme which has tremendous medical importance for use in dissolving blood clots.
  • tPA is a serine protease which catalyzes the conversion of plasminogen to plasmin, one of the essential steps in the fibrinolytic cascade. This enzyme is produced naturally by many mammalian cell types, and was purified initially from human uterus (Rijken, D.C. et al. Biochim Biophys Acta (1979) 580: 140-153) and from the Bowes melanoma cell line (Rijken, D.C. et al. J Biol Chem (1981) 256:7035-7041).
  • tPA can be extracted from a variety of tissues and has been purified from pig heart (Wallen, P. et al. Biochim Biophys Acta (1982) 719: 318-328). A recent review of tPA and its characterization is found in Klausner, A. Biotechnology (1986) 4:706-71.
  • An international standard of human tPA having an activity of 1,000 units per vial has been prepared and is used as the primary standard in assessing tPA activity.
  • This standard purified tPA was prepared from a Bowes melanoma cell conditioned media and purified to a specific activity of about 500 ⁇ 10 3 IU/mg.
  • assays are available for tPA, and the international unit is defined in terms of a comparison of the tested preparation to the activity shown by this primary standard in the same assay.
  • the apparent activity of the preparation will vary in absolute terms, but not relative to the standard, depending on the precise conditions of the assay.
  • Verheijen, J.H., et al., Thrombosis Research (1982) 27:377-385 several factors are known to influence activity. This assay involves the catalyzed conversion of plasminogen to plasmin and measurement of the plasmin formed colorimetrically.
  • tPA preparations in this assay can be enhanced by addition of cyanogen bromide fragments of fibrinogen, by the addition of fibrinogen itself, or by the addition of fibrin.
  • Other useful in vitro assay systems include the direct measurement of lysis of fibrin clots as described by Wallen, P. 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 Sepharose. 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.
  • Type I and Type II tPA have been reported as partially separated on arginine-Sepharose or lysine Sepharose.
  • Ranby, M. et al. FEBS Letters (1982) 146:239-242 used arginine-Sepharose and obtained two overlapping peaks of unreported specific activity. Ranby also showed the differences in molecular weights in these two peaks to reside in the A chain, and cited the observation of Komanger, C. et al. Thrombosis and Hematosis (1981) 46:602-603 that these two types occur in a variety of mammalian tissues.
  • Thrombosis and Hemostasis (1985) 54: 788-791 reported the separation of the two types of tPA on lysine Sepharose and concluded from comparison studies with tPA preparations which had been secreted in the presence of tunicamycin, an inhibitor or N-glycosylation, that glycosylation at the asparagine residues had no influence on the enzymatic activity of tPA.
  • Einarsson, M. et al. Biochim Biophys Acta (1985) 830: 1-10 reported separation of Type I and Type II on lysine Sepharose. Although their data showed only partial separation, they indicated that lengthening the column permitted complete separation of the two forms. Their results were asserted to be superior to those reported earlier by Ranby. Specific activities in the range of 150 ⁇ 10 3 IU/mg for Type I and 225 ⁇ 10 3 IU/mg for Type II were obtained using the World Health Organization International reference preparation of urokinase (established 1975, 66, 46).
  • Type I and Type II tPA have differential activities according to the conditions of the assays employed, it is apparent that the spectrum of behavior to be exhibited in vivo by each of these molecules will differ. Accordingly, it is advantageous to obtain highly purified forms of each of these two biologically distinct types.
  • Type I and Type II tPA in separate and highly purified preparations.
  • the method of the invention makes available to the art each of these variants in a highly homogeneous and active form. Accordingly, in one aspect, the invention is directed to a method to purify Type I and Type II tPA to a specific activity of the order of 500 ⁇ 10 3 IU/mg, when measured in the fibrin-stimulated rate assay described hereinbelow using CHO derived recombinant tPA. These values are those obtained for CHO-derived tPA using the WHO International tPA standard.
  • the method comprises first, subjecting conditioned media from cells cultured to produce tPA to rapid temperature lowering to the order of 4oC; purifying the tPA from the cooled media by a combination of ion exchange chromatography and affinity chromatography, and then separating the Type I and Type II variants on a dimensionally effective lysine Sepharose column.
  • the separated Type I and Type II proteins are then recovered using a gel filtration technique and re-constituted in solubilizing buffer for formulation.
  • the invention is directed to Type I tPA preparation having a specific activity corresponding to more than about 420 ⁇ 10 3 IU/mg for CHO-derived tPA, and to a Type II preparation of tPA having a specific activity corresponding to more than about 550 ⁇ 10 3 IU/mg for CHO-derived tPA, both as measured in the fibrin-stimulated rate assay described below.
  • the invention is directed to pharmaceutical compositions containing the highly purified, isolated Type I or Type II tPA, and to methods of dissolving blood clots and ameliorating the symptoms of heart attacks in patients utilizing these compositions.
  • Figure 1 shows a schematic diagram for purification of tPA from conditioned media prior to application to lysine Sepharose.
  • Figure 2 shows the elution of tPA from Fast-S Sepharose.
  • Figure 3 shows the elution pattern of recombinantly produced tPA from a PABA column.
  • Figure 4 shows the elution of Type I and Type II tPA from lysine Sepharose.
  • Figure 5 shows the elution pattern of native tPA from an immunoaffinity column.
  • 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 contains 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 117 and 448 of the Pennica structure.
  • 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 lyophilized preparetion of tPA Type I and Type II unseparated, having a total of 1000 units of activity 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.
  • the specific activity relative to the standard will not change by virtue of this variation. That is, 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.
  • Specific activity corresponding to the defined activity for CHO-derived tPA refer to a tPA having a degree of purity corresponding to that of recombinant CHO-derived tPA which gives the specified activity in the assays.
  • the specific number obtained even with respect to the same tPA standard will vary with the origin of the tPA due to post-translational processing alterations which differ from host to host.
  • the specific activity of 100% pure, homogeneous tPA Type I for example, will be different depending on whether this is isolated from CHO cells or colon cells. That the candidate tPA has the same level of purity of CHO-derived defined specific activity can be ascertained by comparative analysis of the two samples on analytical HPLC and SDS-PAGE.
  • a particular tPA sample will have a specific activity corresponding to that greater than 420 ⁇ 10 3 IU/mg of CHO-derived tPA if the level of impurities in this sample is less than that found in a tPA derived from CHO cells, which has the specific activity noted.
  • This can be ascertained by conducting SDS-PAGE under standard conditions, both for the sample and the CHO-derived tPA, and estimating the percentage of impurities in each sample. SDS-PAGE is more sensitive to levels of impurities than HPLC, and is the primary test method.
  • the sample in questions shows a level of 1% impurities on SDS-PAGE, and the CHO-derived tPA of specific activity 420 ⁇ 10 3 IU/mg shows 1.5% impurities by this same method, the sample will have been shown to have a specific activity by this definition greater than that corresponding to 420 ⁇ 10 3 IU/mg for CHO-derived tPA. At this level of purity, both samples would be expected to show a single peak in analytical HPLC.
  • a convenient assay used in defining the above parameters is the fibrin-stimulated microtiter tPA assay.
  • plasminogen is converted to plasmin by tPA in the presence of fibrin as a stimulator, and the plasmin formed is assayed using the synthetic substrate S-2251.
  • Immulon II 96-well microtiter plates are used. Into each well are placed 200 microliters of 10 mcg/ml solution in water of fibrinogen (from which plasminogen had been removed by lysine Sepharose column). The plates are allowed to air dry overnight.
  • test plate is incubated for 15 minutes at 37oC, and the reaction is started by the addition of 100 microliters of a plasminogen/S-2251 mixture which contains two parts 10 mM S-2251, one part plasminogen solution (1 mcg/ml) and seven parts assay buffer.
  • the mixture is prepared immediately prior to use.
  • Assay buffer is Tris saline prepared by dissolving 605 mg Tris in 800 ml distilled water and adjusting the pH to 8.8 with 6 normal HCl, then dissolving 5.84 grams NaCl and 0.1 grams Triton X-100 into the buffer and diluting to 1,000 ml.
  • the absorbance (405 nm) is then measured at 20, 40 and 60 minutes, and standard curves are plotted using absorbance values of 1 or less.
  • the tPA standard is the MB-1022 standard using dilutions of 0-50 IU/ml.
  • Type I and Type II tPA through separation of a highly purified tPA preparation. Attainment of the high specific activity starting material is attributable to a combination of efficient purification techniques which include immediate recovery to a lower temperature of conditioned medium producing tPA, removal of the tPA from the medium by ion exchange, and an additional purification step utilizing an affinity support. Detergent is not used in any of the steps.
  • the conditioned media are brought from the elevated temperature at which the cells are metabolizing to about 4oC and the pH lowered to 5.5 with dilute acetic acid.
  • the media is then applied to a column of Fast-S Sepharose which had been previously equilibrated with 50 mM NaOAc/100 mM NaCl, pH 5.5 to adsorb the tPA.
  • Other cation exchange resins may also be used.
  • the method of the preferred embodiment diverges for recombinantly produced and native tPA.
  • the adsorbed tPA is eluted with 20 mM Tris/1 M NaCl pH 8.0 and the eluate applied to a P-aminobenzamidine agarose (PABA) affinity resin which had been pre-equilibrated with 20 mM Tris/1 M NaCl, pH 8.0.
  • PABA P-aminobenzamidine agarose
  • the adsorbed tPA is eluted from the PABA column with 20 mM Tris/1 M NaCl/2.5 M KSCN, pH 7.0 and diluted with 2 volumes of 20 mM Tris, pH 7.0.
  • the diluted eluate is then precipitated with ammonium sulfate at approximately 300 g/l to precipitate the tPA.
  • the adsorbed PA can be eluted from the PABA column with 20 mM Tris, pH 7.0, 1 M NaCl, and 1 M arginine-HCl, and then ammonium sulfate precipitated without prior dilution.
  • the precipitate is then dissolved with 150 mM arginine hydrochloride/50 mM sodium citrate, pH 6 and subjected to gel filtration on Sephacryl S-300 pre-equilibrated with the same formulation mixture.
  • the resulting tPA has a specific activity of approximately 5 ⁇ 10 5 IU/mg.
  • the elution from Fast-S Sepharose is conducted in the presence of 20 mM Tris/1 M NaCl/150 mM arginine hydrochloride, pH 7, and the eluted material is applied to an immunoaffinity column containing mouse derived anti tPA antibodies which is pre-equilibrated in the same buffer.
  • the column is washed with the same buffer mixture but in the presence of 1 M KSCN, and the tPA is then eluted by increasing the concentration of KSCN to 3 M.
  • the eluate from the immunoaffinity column is then concentrated 10-20 fold and diluted with 2 volumes of 20 mM Tris pH 7, and the material precipitated with am monium sulfate at 300 g/1 as for the recombinant material.
  • the precipitated tPA/ammonium sulfate mixture was then dissolved in 1 M arginine hydrochloride, pH 6, and applied to gel filtration with Sephacryl S-300 using the same formulation.
  • a clean separation into Type I and Type II tPA can be made using an appropriately dimensioned column of Sepharose conjugated to a basic amino acid, preferably a lysine Sepharose column.
  • a suitable column can be constructed at a volume of 2.6 ⁇ 100 cm which column is surprisingly efficient and effective in obtaining complete separation of Type I and Type II tPA over a ten fold difference in total tPA loaded onto the column.
  • the column of this dimension is as effective in separating 700 mg of the mixture into its respective Type I and Type II components as it is in resolving only about 70 mg. Accordingly, this surprising result permits the preparation of large amounts of homogeneous and highly purified tPA I and tPA II of high specific activity in a single step on a practical scale.
  • Example 1 Purification of recombinant tPA expressed in CHO cells.
  • the purification scheme used for CHO-tPA is presented in Figure 1 and described below in steps 1-3.
  • the buffers referred to are as follows:
  • #5 50 mM Sodium Citrate; 150 mM Arginine-HCl, pH 6.0.; #6: PBS, pH 8.0, 0.1 M KSCN;
  • Step 1 A 5-liter S-Sepharose Fast-Flow bioprocess column (Pharmacia; 25.2 cm x 10 cm) was equilibrated with five column volumes of Buffer #1 at 150 1/h. Cell culture conditioned media are pH adjusted to 5.5 with dilute acetic acid. After adjusting, the media are then loaded onto the column at the same flow rate. The pH and conductivity of the media were such that the tPA bound selectively to the column electrostatically while the bulk of the serum proteins and non-proteinaceous material (i.e. nucleic acids, phospholipids, etc.) did not bind to the resin and were removed in the column flowthrough. The tPA was then eluted from the column with approximately 4 column volumes of Buffer #2. The elution pattern obtained on a much smaller column, run under the same, but scaled-down, conditions, is shown in Figure 2. The elution pattern is identical to that obtained for the procedure described here.
  • Step 2 A 4 liter bioprocess column (25.2 cm ⁇ 8 cm) with p-Aminobenzamidine (PABA) Agarose resin was equilibrated with five column volumes of buffer #2 at 50 1/h.
  • the step 1 elution is pH adjusted to 8-0 with dilute sodium hydroxide and then loaded onto the PABA column at 50 1/h.
  • the tPA was eluted from this column with one-half column volume of buffer #3 at 25 1/h.
  • the elution pattern obtained on a much smaller column, run under the same, but scaled-down, conditions, is shown in Figure 3.
  • the elution pattern is identical to that obtained for the procedure described here.
  • Step 3 The elution from step 2 was diluted
  • the ammonium sulfate pellets are thawed at 4oC and resolubilized in a restricted volume of Buffer #5 at a concentration of 15-20 mg/ml.
  • the solution was applied to a 10-liter Sephacryl S-300 bioprocess column (Pharmacia; 11.3 cm ⁇ 100 cm) previously equilibrated with approximately two column volumes of Buffer #5. Elution fractions (approximately 150 ml each) from the column were analyzed by SDS-PAGE and enzymatic assay. The fractions containing "active" and pure (>95%) material were pooled.
  • Step 4 Separation of Type I and Type II tPA: The purified tPA obtained from step 3 was then separated cleanly into Type I and Type II tPA using a 2.6 ⁇ 100 cm column of lysine Sepharose to selectively adsorb the tPA. The column is pre-equilibrated with buffer #6 and the tPA loaded onto the column in the various milligram amounts shown in Table 1. Elution was effected by application of a gradient of buffer #6 and #7 which resulted in a clean
  • Type I tPA shows an activity of 420 ⁇ 10 3 IU/mg, within experimental error; that eluted as Type II tPA shows a specific activity of about 550 ⁇ 10 3 IU/mg within experimental error.
  • Example 2 Preparation of Type I and Type II tPA from Colon Cells Colon cells (CD 18 Co, ATCC CRL 1459) which normally secrete tPA are cultured and the supernatant harvested. The supernatant is subjected to purification steps as described above except that in place of the PABA affinity column of Step 2, an immunoaffinity column using antiserum raised in response to immunization of rabbits with the purified CHO-derived tPA of Example 1 is used. The elution pattern is shown in Figure 5.

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Abstract

Les tPA (activateur plasminogène de tissu) de type I et de type II ont une configuration de glycolsylation et une activité biologique quantitative qui différent. On peut obtenir ces deux formes de tPA sous forme très purifiée grâce au procédé décrit dans cette invention.
PCT/US1989/001406 1988-04-07 1989-04-04 tPA DE TYPE I ET DE TYPE II PURIFIE WO1989009820A1 (fr)

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US178,392 1988-04-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0493037A2 (fr) * 1990-12-24 1992-07-01 Eli Lilly And Company Méthode de traitement des troubles thromboemboliques
CN113337490A (zh) * 2021-06-18 2021-09-03 广州铭康生物工程有限公司 一种大规模快速分离纯化rhTNK-tPAI/II型的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 23, issued 1984, G. POHL: "Tissue Plasminogen Activator: Peptide Analyses Confirm an Indirectly Derived Amino Acid Sequence, Identify the Active Acid Serine Residue, Establish Glycosylation Sites, and Localize Variant Differences", see pages 3701-3707. *
BIOCHIM. BIOPHYS. ACTA, Volume 830, issued 1985, M. EINARSSON: "Large scale Purification of Human-type Plasminogen Activator Using Monoclonal Antibodies", see pages 1-10. *
EUR. J. BIOCHEM., Volume 132, issued 1983, P. WALLEN: "Purification and Characterization of Melanoma Cell Plasminogen Activator", see pages 681-686. *
FEBS LETT, Volume 209, No. 1, issued December 1986, I. Dodd: "Large Scale Purification of Recombinant Tissuetype Plasminogen Activator", see pages 13-17. *
FEBS LETT., Volume 146, No. 2, issued September 1982, M. RANBY, "Isolation of Two Variants of Native One-chain Tissue Plasminogen Activator", see pages 289-292. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0493037A2 (fr) * 1990-12-24 1992-07-01 Eli Lilly And Company Méthode de traitement des troubles thromboemboliques
EP0493037A3 (en) * 1990-12-24 1992-09-09 Eli Lilly And Company Method of treating thromboembolic disorders
CN113337490A (zh) * 2021-06-18 2021-09-03 广州铭康生物工程有限公司 一种大规模快速分离纯化rhTNK-tPAI/II型的方法

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