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EP0275282A1 - Procede de purification d'un activateur de plasminogene - Google Patents

Procede de purification d'un activateur de plasminogene

Info

Publication number
EP0275282A1
EP0275282A1 EP87904701A EP87904701A EP0275282A1 EP 0275282 A1 EP0275282 A1 EP 0275282A1 EP 87904701 A EP87904701 A EP 87904701A EP 87904701 A EP87904701 A EP 87904701A EP 0275282 A1 EP0275282 A1 EP 0275282A1
Authority
EP
European Patent Office
Prior art keywords
tpa
buffer
acm
mother solution
chromatography medium
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Withdrawn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
EP87904701A
Other languages
German (de)
English (en)
Inventor
Andrew Charles Kenney
Angela Joyce Perry
Susan White
Fiona Angela Olinda Marston
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.)
Celltech R&D Ltd
Original Assignee
Celltech Ltd
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
Priority claimed from GB868617394A external-priority patent/GB8617394D0/en
Priority claimed from GB878709288A external-priority patent/GB8709288D0/en
Application filed by Celltech Ltd filed Critical Celltech Ltd
Publication of EP0275282A1 publication Critical patent/EP0275282A1/fr
Withdrawn legal-status Critical Current

Links

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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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 present invention relates to a purification process, and in particular to a process for purifying human tissue plasminogen activator (tPA). It is envisaged that the process will be of particular, but not exclusive, application in the purification of tPA produced by recombinant DNA technology.
  • tPA is an enzyme (and therefore a protein) which is found in the blood and various tissues of the body. It is involved in the processes in the body whereby an intact blood circulation system is maintained. In particular, tPA is involved in the regulation of blood clot formation.
  • a blood clot is formed by laying down a mass of fibrin which forms the basis of the clot.
  • the fibrin has associated with it an enzyme precursor, plasminogen.
  • Plasminogen can be activated to produce the enzyme plasmin which acts on the fibrin in the blood clot to cause dissolution of the clot (thrombolysis) .
  • streptokinase which is an enzyme derived from various streptococcus strains. Streptokinase has been used as a thrombolytic agent, but has the disadvantages that it activates plasminogen whether it is circulating or bound in a clot and that it is antigenic. The first disadvantage can lead to uncontrolled bleeding in parts of the body remote from the targetted clot, and the second disadvantage can lead to adverse immunological reactions in the body which can lead fccc as. reduction in the efficiency of the action of the streptokinase or to undesirable clinical symptoms.
  • the second plasminogen activator is generally known as urokinase since it was originally isolated from human urine. More recently, it has been isolated from tissue culture systems or has been produced by recombinant DNA technology. Since urokinase is of human origin, it is not antigenic. - However, it has the disadvantage that it activates both circulating and bound plasminogen, leading to the problem set out above.
  • tPA The third plasminogen activator is tPA which is also non-antigenic in humans. Moreover tPA is very specifically bound to fibrin and is therefore able to act almost exclusively on plasminogen bound into a blood clot. Thus, tPA has the advantage that it is a highly specific thrombolytic agent. It has already been used clinically for the treatment of thrombi in patients who have received a kidney transplant (see Weimar et al. , The Lancet, 7th November, 1981, pages 1018 and 1019)
  • tPA could only be isolated in small quantities from human tissues, such as human uterine tissue.
  • human tissues such as human uterine tissue.
  • tPA should now be available in commercially viable quantities.
  • tPA should now be available in commercially viable quantities.
  • tPA produced by melanoma cell culture it will be necessary to separate it from the components of the culture medium and such other human proteins as are normally produced by the melanoma cells.
  • tPA produced by recombinant DNA technology
  • tPA as it is normally produced, for instance by the melanoma cell line or by recombinant DNA technology, is a one chain protein. However, it is subsequently cleaved into a two chain, disulphide-linked protein. This cleavage can occur spontaneously during handling of the tPA following its production.
  • the one chain form is more efficacious in clearing blockages in arteries (see Garabedian, H.D. et al. , JACC, , 3, 599-607, 1987). It is preferred therefore that the tPA as purified should be in the one chain form so that, after purification, it can be used in the one chain form.
  • Both one chain and two chain molecules can each be found in two variants which differ in molecular weight by about 3,000 Daltons. This difference is probably due to different degrees of glycosylation at one (Asn-184) of four potential glycosylation recognition sites in the molecule.
  • both one chain forms of tPA contain N-terminal sequence variants, (see Jornvall, H. et al., FEBS Letters, 156, 1, 47-50, 1983).
  • N-terminal sequence variants see Jornvall, H. et al., FEBS Letters, 156, 1, 47-50, 1983.
  • tPA should be purified by affinity chromatography using an anti-tPA monoclonal antibody bound to a chromatography medium such as Sepharose 4B.
  • a chromatography medium such as Sepharose 4B.
  • any equilibration or elution buffers are given. It is envisaged that, for instance, supernatant from a melanoma cell line culture could be treated by such a process to produce purified tPA. It is indicated that the yield of tPA is as much as 90%, but no indication of the purity of the tPA, nor of the ratio of one to two-chain types, is given.
  • tPA is generally highly adherent to vessels, in particular glass vessels, in which it is stored or through which it .is passed. There can therefore be a considerable loss of material during processing. Moreover, it is generally difficult to maintain a high concentration of pure tPA in solution. At a concentration of about 1 mg/ml, the tPA tends to precipitate out. This makes it less useful in the therapeutic field, where a high concentration solution is desirable.
  • tPA could be kept in solution by the addition of auxiliary agents, such as IM ammonium bicarbonate, lysine, arginine, other amino acids or other amines. These are also considered to be active in maintaining the stability of the tPA.
  • auxiliary agents such as IM ammonium bicarbonate, lysine, arginine, other amino acids or other amines.
  • auxiliaries may not be acceptable in a therapeutic context. It would therefore be desirable to produce a high concentration, stable tPA solution without needing to use any possibly unacceptable auxiliaries.
  • a process for the purification of tPA comprising carrying out a) an ion exchange chromatography step on a highly anionic chromatography medium and b) an affinity chromatography step on a mother solution containing tPA.
  • all the purification steps are carried out at as low a pH as possible without adversely affecting the tPA or the properties of the chromatography media used.
  • the pH should not fall below 4. It has surprisingly been found that at such low pHs, the tPA is both storage stable and able to stay in solution at concentrations well above lmg/ l.
  • At least the later purification steps, and preferably the whole purification procedure is carried out in the presence of about 0.01% of a detergent, preferably a non-ionic detergent.
  • a detergent preferably a non-ionic detergent.
  • a particularly suitable detergent is Tween R 80, which is known to be therapeutically acceptable. Such detergents appear to reduce the amount of tPA adhering to the purification or storage vessels.
  • the process comprises: applying the mother solution containing tPA to a highly anionic chromatography medium equilibrated with a buffer at 25 mS ionic strength or lower and at a pH between 4 and 7; eluting the tPA from the highly anionic chromatography medium with a high ionic strength buffer; applying the eluate from the highly anionic chromatography medium to an affinity chromatography medium, comprising an anti-tPA antibody covalently bound to a chromatography material, equilibrated with a buffer which does not substantially interfere with the binding of the tPA to the antibody; and eluting the tPA from the affinity chromatography medium with a buffer which disrupts the binding of the antibody to the tPA.
  • the mother solution containing the tPA may be the supernatant from a culture of tPA secreting cells, such as Bowes melanoma cells.
  • the mother solution may be the supernatant from a culture of a eukaryotic cell line which has been transformed by recombinant DNA technology so that it is able to express and secrete tPA.
  • the mother solution if necessary, may be clarified to remove any cells ⁇ r cell debris.
  • the clarification may be carried out, for instance, by centrifugatiori or filtration.
  • the clarification should not be carried out by any process, such as ultrafiltration, which could adversely affect the yield of tPA.
  • Ultrafiltration in particular has been found to reduce significantly the yield of tPA.
  • the mother solution is clarified, where necessary, by filtration, for instance using a hydrophilic resin-impregnated glass-fibre filter, such as a Balston LP-200-50-80 cartridge filter (which is a polypropylene and borosilicate glass-fibre depth filter having a 0.22 urn nominal pore size) .
  • a hydrophilic resin-impregnated glass-fibre filter such as a Balston LP-200-50-80 cartridge filter (which is a polypropylene and borosilicate glass-fibre depth filter having a 0.22 urn nominal pore size) .
  • the solution in which the cells are cultured to produce the mother solution contains a serine protease inhibitor, such as aprotinin, to inhibit any enzymatic cleavage of the tPA. If such an inhibitor is used, it will be necessary to maintain the pH in the mother solution and in at least the initial purification steps above 4 so that the inhibitor remains active. It has been found that a preferred level of aprotinin for obtaining one chain tPA is from 10 to 100 KlU/ml of mother solution.
  • the highly anionic chromatography medium is of the rigid type so that fast flow rates without compaction of the medium can be achieved.
  • Rigid HACMs are well known in the art.
  • the anionic groups on the HACM may be any of those known in the art, such as carbox late, sulphate, sulphonate, sulphopropyl or phosphate groups.
  • the anionic groups are sulphonate groups.
  • a particularly suitable HACM is S-Sepharose Fast Flow (sold by Pharmacia Ltd.), which is a rigid medium comprising a highly cross-linked beaded agarose matrix having attached thereto -CH2 ⁇ S ⁇ 3 a (sulphonate) groups.
  • the HACM is washed with equilibration buffer following application of the (clarified) mother solution.
  • the pH of the HACM equilibration buffer is preferably between 4 and 7 and is preferably about 6 or lower. It has been found that if the pH is too low, undesired proteins become attached to the medium, whereas if the pH is too high, the t?A does not become attached to the medium in sufficient quantity.
  • the ionic strength of the HACM equilibration buffer should be adjusted to suit the pH used. For high pHs, low ionic strengths should be used. For instance, at pH 6.2 an ionic strength not above 10 mS is suitable. At a pH of 4, the ionic strength could rise to about 25mS.
  • the buffer used to equilibrate the HACM comprises 50 mM acetate, 0.01% Tween 80 adjusted to pH 6.0.
  • buffering agents such as phosp.hate or citrate buffers or mixtures thereof
  • ionic strength adjusting agents such as sodium or potassium chloride
  • the high ionic strength buffer used to elute the tPA from the HACM comprises 50 mM * acetate, 0.01% Tween 80 and 500 mM sodium chloride adjusted to pH 6.0.
  • the ionic strength of the HACM elution buffer is not less than that of a solution containing 50 mM acetate and 200 mM, preferably 250 mM, sodium chloride adjusted to pH 6.0, and is advantageously the same as or greater than that of the preferred elution buffer. However, it should be ensured that the ionic strength of the buffer is not so high as to affect adversely the tPA.
  • the affinity chromatography medium preferably comprises a monoclonal anti-tPA antibody, for instance of the type disclosed in DE-A-3 222 084, covalently bound to a suitable chromatography material.
  • the antibody may comprise a polyclonal anti-tPA antibody.
  • Preparation of ACMs is well known in the art and any one of these known methods may be used.
  • the anti-tPA antibody is coupled to the chromatography material by use of cyanogen bromide activation, for instance as described in Affinity Chromatography, Principles and Methods, pages 11 to 18, by Pharmacia Ltd. (and available from them).
  • Chromatography materials suitable for the preparation of ACMs are well known in the art and a suitable one can readily be selected by those skilled in the art.
  • the chromatography material is rigid, for the reasons set out above in relation to the HACM.
  • a particularly suitable chromatography material for producing the ACM is Sepharose CL4B (sold by Pharmacia Ltd.) which is a semi-rigid material comprising a cross-linked agarose polymer.
  • the ACM is washed with equilibration buffer after the application thereto of the HACM eluate.
  • the pH and ionic strength of the ACM equilibration buffer should be adjusted to facilitate the binding of the tPA to the antibody, and in particular the ionic strength should not be so great or so low as to disturb such binding.
  • the pH of the ACM equilibration buffer is between 5 and 10, and is advantageously between 5 and 7 and most preferably is about 5.5.
  • the ACM equilibration buffer is preferably of high ionic strength, for instance, equivalent to or greater than that of a solution containing lOOmM sodium chloride, since in some cases this reduces the incidence of non-specific binding of undesired protein to the ACM.
  • the ACM equilibration buffer contains 100 mM acetate, 200mM sodium chloride and 0.01% Tween 80 adjusted to pH 5.5.
  • the ACM elution buffer should be able readily to disrupt the binding of the tPA to the antibody without adversely affecting either.
  • the buffer may have an extremely high or an extremely low pH or ionic strength.
  • the buffer may contain a chaotropic salt, such as magnesium chloride, potassium thiocyanate or potassium iodide, or a protein denaturing agent, such as urea or guanidine.
  • the buffer may contain a disruptive solvent, such as propanol or ethylene glycol.
  • the ACM elution buffer is a low pH buffer, for instance containing 50 mM acetate, 0.01% Tween 80 and 200 mM sodium chloride adjusted to pH 3.5.
  • the use of such low pHs does not present any problems as regards the stability of the tPA.
  • the ACM elution buffer contains a chaotropic salt, and ma ' y, for instance, contain 5M magnesium chloride, and 20 mM Tris adjus-ted to pH 6.2 with hydrochloric acidfeel
  • variations in the buffering agents and ionic strength adjusting agents may be made in the ACM elution buffer.
  • the ACM eluate is buffer-exchanged using a gel filtration chromatography medium having a low molecular weight exclusion limit.
  • a gel filtration chromatography medium having a low molecular weight exclusion limit.
  • a particularly suitable such medium is Sephadex G-25 (sold by Pharmacia Ltd).
  • the gel filtration chromatography medium is equilibrated with 50mM acetate, 0.01% Tween 80 adjusted to pH 4.0.
  • the eluate from the gel filtration step is concentrated, preferably using a membrane concentration system, such as a Millipore Minitan system.
  • the buffer exchange step may be used to put the tPA into a buffer suitable for intravenous administration, suitable for freezing, or suitable for freeze drying. Since the present process can be carried out speedily and without substantially degrading the tPA, it can be carried out at room temperature. This is advantageous over prior art processes, which are usually carried out at 4°C, since it avoids problems of increased solution viscosities and increased sterilizing times.
  • tPA tPA
  • a second aspect of the present invention is the provision of a stable, concentrated tPA solution comprising the tPA described above in a low pH buffer, which preferably contains a detergent.
  • Figure 1 shows a series of sodium dodecyl sulphate (SDS) 7 to 15% polyacrylamide gradient gels used to monitor the progress of the purification wherein: lane 1 shows molecular weight markers; lane 2 shows mother solution; lane 3 shows eluate from the HACM; lanes 4 and 5 show eluates from the ACM; lanes 6 to 9 show eluates from a buffer exchange column; and lanes 10 to 13 show eluates from a second buffer exchange column; and Figure 2 shows a series of SDS 7 to 15% polyacrylamide gels used to show the purity of the product, wherein: lanes 1 and 5 are molecular weight markers, and lanes 2, 3 and 4 are the eluate from a buffer exchange column at loadings of 6, 12 and 18 micrograms respectively.
  • SDS sodium dodecyl sulphate
  • the samples were reduced and alkylated before application to the gels.
  • the loading was 15 micrograms of protein per lane.
  • the ACM purification step was carried out in two stages and the buffer • exchange was carried out in four stages, as shown in lanes 6 to 9.
  • the buffer exchanged material was then concentrated in four stages on a Millipore Minitan system, as shown in lanes. 10 to 13.
  • a rat cell line which is able to express and secrete tPA was produced by recombinant DNA technology in a manner similar to that described in GB-B-2 119 804. This was cultured in a standard culture medium containing 50 KlU/ml aprotinin in a fermenter to produce a mother solution containing tPA and aprotinin.
  • the column was eluted using a buffer containing 50 mM acetate, 0.01% Tween 80 and 500 mM sodium chloride adjusted to pH 6.0 until elution of the tPA was complete.
  • the eluate from the S-Sepharose column was immediately applied to the affinity chromatography column, which was then washed with the affinity chromatography equilibration buffer until no more protein was eluted.
  • the tPA was then eluted from the column by use of a buffer containing 50 mM acetate, 0.01% Tween 80 200mM sodium chloride adjusted to pH 3.5 acid until all the protein had been eluted.
  • the affinity chromatography eluate contained 86% of the applied tPA, which was substantially pure and which had a specific activity of greater than 100 x 10 3 ⁇ /mg.
  • the affinity chromatography eluate was applied to a 2.2 1 Sephadex G25-M gel filtration column equilibrated with 50mM acetate, 0.01% Tween 80 adjusted to pH 4.0.
  • the tPA was eluted in the buffer free of the components of the ACM elution buffer, which were retained on the column.
  • the collected buffer contained 88% of the applied tPA which was substantially pure and which had a specific activity of 110 x 10 3 U/mg.
  • the tPA was further concentrated by application to a Millipore Minitan system. It can thus be seen that the process of the present invention produced a quantity of the tPA which is substantially pure in a simple operation carried out at room temperature.
  • the overall yield of the process was 67%
  • the process can readily be operated on a scale sufficient to produce commercially viable quantities of tPA.
  • this solution containing the tPA at a concentration in excess of Img/ml in a low pH buffer containing a detergent, did not form precipitates, was storage stable and did not enable the tPA to adhere to the walls of the container.
  • N-terminal analysis of the product of this process showed that 94% of it had the N-terminal sequence.
  • H2-G-A-R-S-Y-Q-V-I that is, the full amino acid sequence
  • the product tPA was separated according to a conventional procedure at conventionally used pHs and ionic strengths. No special precautions were taken to carry out the purification as fast as possible.
  • the product from this control run consisted essentially of the one chain form of tPA. However, it had a variety of species having different N-terminal sequences. The following distribution was observed: NH 2 -G-A-R-S-Y-Q-V-I 58 %

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Abstract

Un procédé de purification de tPA comprend: l'application d'une solution mère contenant du tPA sur un milieu chromatographique très anionique (HACM) maintenu par un tampon à une puissance ionique équilibrée égale ou inférieure à 25mS et à un pH compris entre 4 et 7; l'élution du tPA du milieu chromatographique très anionique avec un tampon à puissance ionique élevée; l'application de l'éluat du milieu chromatographique très anionique sur un milieu de chromatographie par affinité (ACM) comprenant un anticorps anti-tPA lié par covalence à une substance chromatographique maintenue par un tampon à un pH équilibré compris entre 5 et 10; et l'élution du tPA du milieu de chromatographie par affinité avec un tampon qui rompt la liaison entre l'anticorps et le tPA. De préférence, la solution mère est le surnageant de culture d'une lignée de cellules eukaryotes transformées par la technique de l'ADN recombinant de façon à devenir capables d'exprimer et de sécréter du tPA. La solution mère est avantageusement traitée dès sa récolte et toutes les étapes du traitement sont effectuées aussi rapidement que possible. La solution mère contient un inhibiteur de la protéase de sérine. Toutes les solutions contiennent un détergent et sont maintenues à un pH aussi bas que possible.
EP87904701A 1986-07-16 1987-07-15 Procede de purification d'un activateur de plasminogene Withdrawn EP0275282A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB8617394 1986-07-16
GB868617394A GB8617394D0 (en) 1986-07-16 1986-07-16 Purification process
GB878709288A GB8709288D0 (en) 1987-04-16 1987-04-16 Purification process
GB8709288 1987-04-16

Publications (1)

Publication Number Publication Date
EP0275282A1 true EP0275282A1 (fr) 1988-07-27

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP87904701A Withdrawn EP0275282A1 (fr) 1986-07-16 1987-07-15 Procede de purification d'un activateur de plasminogene

Country Status (5)

Country Link
EP (1) EP0275282A1 (fr)
JP (1) JPH01500563A (fr)
AU (1) AU7709287A (fr)
DK (1) DK140288A (fr)
WO (1) WO1988000615A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4960702A (en) * 1985-09-06 1990-10-02 Codon Methods for recovery of tissue plasminogen activator
JPS6379591A (ja) * 1986-09-22 1988-04-09 Mitsui Toatsu Chem Inc ｔＰＡの精製方法
DE3832898A1 (de) * 1988-09-28 1990-04-12 Boehringer Mannheim Gmbh Praeparat von in prokaryonten exprimiertem plasminogenaktivator
US20090130714A1 (en) * 2007-09-24 2009-05-21 Reliance Life Sciences Pvt.Ltd. Process for purifying recombinanat tissue plasminogen activator (TPA)

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5935B2 (ja) * 1977-06-03 1984-01-05 住友化学工業株式会社 ウロキナ−ゼの精製法
DE3015699C2 (de) * 1979-04-26 1982-07-15 Asahi Kasei Kogyo K.K., Osaka Herstellung eines Plasminogen-Aktivators
WO1984001786A1 (fr) * 1982-10-28 1984-05-10 Beecham Group Plc Derives d'enzymes et leur utilisation dans le traitement de la thrombose
NL191755C (nl) * 1982-10-29 1996-07-02 Mitsui Toatsu Chemicals Plasminogeenactivator en trombolytisch middel.
DE3479814D1 (en) * 1984-06-05 1989-10-26 Asahi Chemical Ind Process for the preparation of a plasminogen activator
FI89723C (fi) * 1984-10-01 1993-11-10 Genzyme Corp Foerfarande foer framstaellning av vaevnadsplasminogenaktivator, kodande rekombinant-dna daerav och en transformanscell
JPS61221128A (ja) * 1985-03-26 1986-10-01 Snow Brand Milk Prod Co Ltd プラスミノ−ゲン活性化因子に対するモノクロ−ナル抗体とその調製方法及び該モノクロ−ナル抗体の使用方法
AU606582B2 (en) * 1985-09-06 1991-02-14 Codon Methods for the recovery of tissue plasminogen activator

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8800615A1 *

Also Published As

Publication number Publication date
DK140288D0 (da) 1988-03-15
JPH01500563A (ja) 1989-03-01
DK140288A (da) 1988-04-28
WO1988000615A1 (fr) 1988-01-28
AU7709287A (en) 1988-02-10

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