DK173568B1 - Process for Preparing a Plasma-Based, Thrombin Coagulable Protein Concentrate and Protein Concentrate - Google Patents
Process for Preparing a Plasma-Based, Thrombin Coagulable Protein Concentrate and Protein Concentrate Download PDFInfo
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- DK173568B1 DK173568B1 DK198804241A DK424188A DK173568B1 DK 173568 B1 DK173568 B1 DK 173568B1 DK 198804241 A DK198804241 A DK 198804241A DK 424188 A DK424188 A DK 424188A DK 173568 B1 DK173568 B1 DK 173568B1
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- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
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Abstract
Description
i DK 173568 B1in DK 173568 B1
Opfindelsen angår en fremgangsmåde til fremstilling af et plasmabaseret thrombinkoagulerbart proteinkoncentrat, der i det væsentlige omfatter fibrinogen og faktor XIII. Opfindelsen angår også ved fremgangsmåden 5 opnåede proteinkoncentrater, der er letopløselige i vandigt medium ved stuetemperatur og kan benyttes til opnåelse af injicerbart fibrinogen eller biologisk lim.The invention relates to a process for preparing a plasma-based thrombin coagulable protein concentrate which comprises substantially fibrinogen and factor XIII. The invention also relates to protein concentrates obtained by the method 5 which are readily soluble in aqueous medium at room temperature and can be used to obtain injectable fibrinogen or biological glue.
Man ved, at injektioner af fibrinogen gør det muligt at behandle hypofibrinogenese eller konstitu-10 tionel afibrinogenese med eller uden blødninger og også syndrom med akut defibrinering med alvorlige blødninger, i forbindelse med ætiologisk behandling, heparin-behandling eller antiflbrinolyse.It is known that injections of fibrinogen make it possible to treat hypofibrinogenesis or constitutional afibrinogenesis with or without haemorrhage and also acute defibrination syndrome with severe bleeding, in connection with etiologic treatment, heparin therapy or antifibrinolysis.
På den anden side gør biologisk lim det muligt 15 at give effektiv hjælp ved visse typer kliniske indgreb såsom hudtransplantationer, sammensyning af nerver eller arterier, en hurtig sårheling eller en hvilken som helst anvendelse, hvor man søger en blodstandsende og/eller bakteriostatisk og/eller æstetisk virkning.On the other hand, biological glue enables effective assistance in certain types of clinical interventions such as skin transplants, nerve or artery jointing, rapid wound healing, or any application seeking an endothelial and / or bacteriostatic and / or aesthetic effect.
20 Det vides, at fibrinogen, der er en vigtig bestanddel i et sådant koncentrat, undergår enzymatisk nedbrydning ved kontakt med thrombin, der er aktiveret af calciumioner. Efter eliminering af fibrinopeptiderne A og B polymeriserer fibrinmonomere og danner spontant opløse-25 ligt fibrin. Tilstedeværelse af faktor XIII i en sådan type produkt bidrager til at stabilisere fibrin ved kovalente bindinger, idet den gør den uopløselig, dvs. modstandsdygtig overfor opløsningsmidler, f.eks. urinstof. Det således stabiliserede fibrin er ud over sin 30 koagulerende virkning mere modstandsdygtig over for fibrinolyse og mekanisk påvirkning.It is known that fibrinogen, which is an important component of such a concentrate, undergoes enzymatic degradation upon contact with thrombin activated by calcium ions. After elimination of the fibrinopeptides A and B, fibrin monomers polymerize and spontaneously form soluble fibrin. The presence of factor XIII in such a type of product helps to stabilize fibrin by covalent bonds, rendering it insoluble, ie. resistant to solvents, e.g. urea. The fibrin thus stabilized, in addition to its coagulant action, is more resistant to fibrinolysis and mechanical action.
Det er nødvendigt til disse forskellige terapeutiske anvendelser at have et produkt til rådighed, der stammer fra plasma og som er let opløseligt og stabilt 35 under de betingelser, hvor det anvendes. Hvad angår biologisk lim er det yderligere nødvendigt, at en sådan 2 DK 173568 B1 efter kontakt med calciumaktiveret thrombin skal være meget klæbende og meget elastisk,It is necessary for these various therapeutic applications to have a plasma derived product which is easily soluble and stable under the conditions in which it is used. In the case of biological glue, it is further necessary that such contact after calcium contact thrombin must be very adhesive and very elastic,
Opnåelsen af sådanne egenskaber står direkte i forbindelse med produktets art og herved til den måde, 5 der benyttes til oprensning af dette fra plasma. Det er derfor gunstigt at besidde en fremgangsmåde til præparering, der uden videre kan anvendes i industriel skala, men også er tilstrækkelig blid til ikke at forringe de biologiske egenskaber af produktet, når det skal 10 benyttes af læger.The attainment of such properties is directly related to the nature of the product and thereby to the manner used to purify it from plasma. It is therefore advantageous to possess a method of preparation which can be readily applied on an industrial scale, but is also sufficiently gentle not to impair the biological properties of the product when it is to be used by physicians.
Arzneim.-Forsch./Drug Res. 34 (1984), 287-290 beskriver biologisk lim ud fra et frysetørret humant fi-brinogenkoncentrat og en human placentafraktion indeholdende faktor XIII.Arzneim.-Forsch./Drug Res. 34 (1984), 287-290 describe biological glue from a freeze-dried human fibrinogen concentrate and a human placental fraction containing factor XIII.
15 EP-A-85 923 beskriver fibrinogenkoncentrater med indhold af stoffer med urinstof- eller guanidinogrup-per, der ved tilsætning af faktor XIII kan benyttes som biologisk lim og også uden faktor XIII planlægges til intravenøs indgivelse som fibrinogenkoncentrat til men-20 nesker.EP-A-85 923 discloses fibrinogen concentrates containing substances with urea or guanidino groups which, by the addition of factor XIII, can be used as biological adhesive and also without factor XIII are planned for intravenous administration as fibrinogen concentrate to humans.
US-A-4,374,061 beskriver bl. a. præparater indeholdende fibrinogen og faktor XIII.US-A-4,374,061 discloses, inter alia. a. Preparations containing fibrinogen and factor XIII.
Endelig kendes lim indeholdende fibrinogen og faktor XIII fra skrifterne FR 2 448 900 og 2 448 901. Pro-25 dukterne opnås fra udfrosset plasma behandlet med en pufferopløsning, der indeholder plaminogeninhibitor-ak-tivator eller plasmininhibitor, der vil findes i limen i frysetørret tilstand. Produkterne har interessante egenskaber, men må tilvejebringes under anvendelse af 30 ret komplicerede fibrinogen-produktionsmetoder, der i henseende til opnåelse af et tilfredsstillende slutprodukt kræver tilsættelse af andre exogene plasmaproteiner såsom faktor XIII, og hvor der ved produktionen skal tilsættes inhibitorer såsom proteaseinhibitorer af 35 dyrisk oprindelse (eksempelvis bovint aprotinin).Finally, adhesives containing fibrinogen and factor XIII are known from FR 2 448 900 and 2 448 901. The products are obtained from frozen plasma treated with a buffer solution containing plaminogen inhibitor activator or plasmin inhibitor which will be present in the adhesive in freeze-dried state. . The products have interesting properties, but must be obtained using 30 rather complicated fibrinogen production methods which, with respect to obtaining a satisfactory final product, require the addition of other exogenous plasma proteins such as factor XIII and in the production of inhibitors such as protease inhibitors of 35 origin (for example, bovine aprotinin).
3 DK 173568 B13 DK 173568 B1
Ydermere solubiliserer de kendte proteinkoncentrater, især sådanne, der anvendes som lim, ikke i et vandigt medium ved stuetemperatur, og de kan endog være svære at solubilisere ved 37eC, hvilket gør det nødven-5 digt, at man anbringer en magnetrørerstang i frysetørringsbeholderen og stiller en magnetomrører til rådighed til opnåelse af en hurtigere solubilisering.Furthermore, the known protein concentrates, especially those used as glue, do not solubilize in an aqueous medium at room temperature, and they may even be difficult to solubilize at 37 ° C, making it necessary to place a magnetic stir bar in the freeze-drying container and place a magnetic stirrer is available to achieve a faster solubilization.
Det vil derfor være ganske nyttigt at kende en simpel fremgangsmåde til fremstilling af biologisk lim, 10 hvorved man uden tilsætning af exogene proteiner opnår en proteinblanding med de ønskede egenskaber for et sådant produkt. Især skal det opnåede koncentrat til anvendelse som lim have et tilfredsstillende Indhold af fibrinogen, faktor XIII og fibronectin, 15 Den omtalte fremgangsmåde skal også uden videre kunne modificeres, så den omfatter et trin med inaktivering af virus. Det opnåede produkt bør solubiliseres i mindre end 10 minutter ved anvendelsestemperaturen {almindeligvis 18-20°C) uden krav på specielt udstyr.Therefore, it will be quite useful to know a simple method for preparing biological glue, 10 whereby without the addition of exogenous proteins a protein mixture having the desired properties of such a product is obtained. In particular, the concentrate obtained for use as glue must have a satisfactory content of fibrinogen, factor XIII and fibronectin. 15 The process described must also be readily modifiable to include a step of inactivating virus. The product obtained should be solubilized for less than 10 minutes at the operating temperature (usually 18-20 ° C) without the need for special equipment.
20 Produktet skal være stabilt i adskillige timer til lettelse af dets anvendelse og til at garantere, at det er klinisk effektivt.The product must be stable for several hours to facilitate its use and to guarantee its clinical efficacy.
Der er nu overraskende blevet fundet en simpel fremgangsmåde til fremstilling af et plasmabaseret 2 5 thrombinkoagulerbart proteinkoncentrat, der i det væsentlige omfatter fibrinogen og faktor XIII, idet dette proteinkoncentrat som frembragt ved simpel fraktionering indeholder mere end 70% koagulerbart fibrinogen i forhold til hele proteinindholdet og en tilfredsstil-30 lende mængde endogen faktor XIII, og idet der hertil anvendes en plasmafraktion, der er tilgængelig fra alle steder, hvor man fraktionerer plasma.Surprisingly, a simple method has been found for preparing a plasma-based thrombin coagulable protein concentrate substantially comprising fibrinogen and factor XIII, as this protein concentrate produced by simple fractionation contains more than 70% coagulable fibrinogen relative to the entire protein content and a satisfactory amount of endogenous factor XIII, utilizing a plasma fraction available from all plasma fractionation sites.
I overenstemmelse hermed er den omhandlede fremgangsmåde ejendommelig ved, at den omfatter følgende 35 trin: 4 DK 173568 B1 a) udfældelse ved 4°C og ved pH-værdien 7,2 af ikke i kulden udfældet plasma med en 10% vandig eth-anolopløsning i 12 til 24 timer; b) fjernelse af overstanden og vask af bundfaldet 5 ved 4°C med 10% vandig ethanol; c) resolubilisering af bundfaldet i nærvær af lysin ved en koncentration svarende til 0,1 til 0,2 g pr. g protein i slutproduktet; d) behandling ifølge kendt teknik til inaktivering 10 af vira med opløsningsmiddel og detergent; e) en anden udfældelse med 10% vandig ethanol; f) fjernelse af overstanden og resolubilisering af bundfaldet i nærvær af lysin som i trin c) , diafiltrering, fyldning på flasker og frysetørring.Accordingly, the process of the invention is characterized in that it comprises the following 35 steps: a) precipitation at 4 ° C and at the pH of 7.2 of plasma not precipitated with a 10% aqueous ethanol solution for 12 to 24 hours; b) removing the supernatant and washing the precipitate 5 at 4 ° C with 10% aqueous ethanol; c) resolubilization of the precipitate in the presence of lysine at a concentration equal to 0.1 to 0.2 g per ml. g of protein in the final product; d) prior art treatment for inactivation of solvent and detergent viruses; e) another precipitate with 10% aqueous ethanol; f) removing the supernatant and resolubilizing the precipitate in the presence of lysine as in step c), diafiltration, bottle filling and freeze drying.
15 Ved en udførelsesform for fremgangsmåden ifølge opfindelsen gennemføres trin e) ved en temperatur mellem 30 og 40°C og en varighed på ca. 90 minutter.In one embodiment of the method according to the invention, step e) is carried out at a temperature between 30 and 40 ° C and a duration of approx. 90 minutes.
Et opfinderisk proteinkoncentrat opnået ifølge denne udførelsesform er ejendommeligt ved, at det pr.An inventive protein concentrate obtained according to this embodiment is peculiar in that
20 g protein indeholder fra 0,70 til 0,75 g fibrinogen, 100 til 250 IE faktor XIII og fra 0,05 til 0,10 g fibronectin.20 g of protein contains from 0.70 to 0.75 g of fibrinogen, 100 to 250 IU of factor XIII and from 0.05 to 0.10 g of fibronectin.
Ved en anden udførelsesform for fremgangsmåden ifølge opfindelsen gennemføres trin e) ved en tempera-25 tur på 4°C og en varighed på ca. 12 timer.In another embodiment of the process according to the invention, step e) is carried out at a temperature of 4 ° C and a duration of approx. 12 hours.
Et opfinderisk proteinkoncentrat opnået ifølge denne anden udførelsesform er ejendommeligt ved, at det pr. g protein indeholder fra 0,90 til 0,95 g fibrinogen, 100 til 250 IE faktor XIII og fra 0,05 til 0,10 g 30 fibronectin.An inventive protein concentrate obtained according to this second embodiment is peculiar in that g of protein contains from 0.90 to 0.95 g of fibrinogen, 100 to 250 IU of factor XIII and from 0.05 to 0.10 g of 30 fibronectin.
Begge de ovennævnte opfinderiske proteinkoncentrater er fordelagtige ved, at de hurtigt ved stuetemperatur solubiliseres i et vandigt medium op til et proteinindhold på ca. 150 g/1.Both of the aforementioned inventive protein concentrates are advantageous in that they are rapidly solubilized at room temperature in an aqueous medium up to a protein content of ca. 150 g / l.
35 5 DK 173568 B135 5 DK 173568 B1
De vil yderligere være stabile efter genopløsning i mindst 24 timer, når de holdes ved en temperatur på f. eks. 4-37°C.They will further be stable after redissolution for at least 24 hours when kept at a temperature of, for example, 4-37 ° C.
Koncentrater fremstillet ved fremgangsmåden ifølge 5 opfindelsen indeholder med fordel pr. g protein mindst 100 IE endogen faktor XIII.Advantageously, concentrates prepared by the process according to the invention contain pr. g protein at least 100 IU endogenous factor XIII.
Yderligere indeholder de en afbalanceret mængde fibronectin, især mellem 0,03-0,10 g/g protein.Furthermore, they contain a balanced amount of fibronectin, especially between 0.03-0.10 g / g protein.
Til anvendelse som injicerbart fibrinogen formu-10 lerer og behandler man koncentratet, så man efter opløsning ved resolubilisering opnår et totalt proteinindhold på ca. 17-29 g/1. Hvis koncentratet skal anvendes som biologisk lim, skal dette indhold være ca. 100-120 g/1.For use as injectable fibrinogen, the concentrate is formulated and treated so that, after dissolution by resolubilization, a total protein content of approx. 17-29 g / l. If the concentrate is to be used as a biological adhesive, this content should be approx. 100-120 g / l.
15 Mere detaljeret kan her som opfinderiske protein koncentrater nævnes et sådant, der er ejendommeligt ved, at dets proteinindhold er indstillet på mellem 17 og 20 g/1 for at gøre det anvendeligt som injicerbart fibrinogen til terapeutisk anvendelse; samt et sådant, 20 der er ejendommeligt ved, at dets protein-indhold er indstillet på mellem 100 og 120 g/1 for at gøre det anvendeligt som biologisk lim ved tilsætning af 500 IE/ml calcisk (d.v.s. calciumaktiveret) thrombin.More specifically, as inventive protein concentrates, mention may be made here of such a property that its protein content is set between 17 and 20 g / l to make it useful as injectable fibrinogen for therapeutic use; and one characterized in that its protein content is set between 100 and 120 g / l to make it useful as a biological adhesive by the addition of 500 IU / ml of calcic (i.e., calcium-activated) thrombin.
Den ovenfor anførte fremgangsmåde ifølge opfindel-25 sen til fremstilling af plasmabaserede, thrombinkoagu-lerbare proteinkoncentrater,der i det væsentlige omfatter fibrinogen og faktor XIII, omfatter et trin med ud-fældelse ved lav temperatur med fortyndet ethanol ved en pH-værdi nær neutralværdien, fra totalt plasma, der 30 ikke underkastes udfældelse ved udfrysning.The above process of the invention for the preparation of plasma-based, thrombin coagulable protein concentrates comprising essentially fibrinogen and Factor XIII comprises a low temperature dilution step with diluted ethanol at a pH near the neutral value. from total plasma that is not subjected to freeze precipitation.
6 DK 173568 B16 DK 173568 B1
Man tilpasser arbejdsbetingelserne ved fremgangsmåden/ så man opnår et bundfald, der kun er lidt denatureret til undgåelse af de nedbrydninger, som proteiner ville blive udsat for, hvis udfældningen tog 5 sted under sædvanlige industrielle betingelser for bearbejdning af plasma. Foruden omhyggelige foranstaltninger, især hvad angår koncentration af ethanol og den anvendte temperatur, bearbejder man foretrukket plasmaet i små beholdere (f.eks. på 10 liter) med magnet- 10 omrørere. Man bringer plasma i kontakt med ethanol af lav koncentration, f.eks. 8-12%, i en tidsperiode, der kan udstrækkes til adskillige dage og i hvert tilfælde er større end 24 timer. Derefter fradekanterer man su-pernatanten, der kan anvendes ved fremstilling af andre 15 derivater.The working conditions of the process are adjusted to obtain a precipitate which is only slightly denatured to avoid the degradation that proteins would be subjected to if the precipitation took place under usual industrial processing conditions for plasma processing. In addition to careful measures, especially regarding the concentration of ethanol and the temperature used, the plasma is preferably processed in small containers (e.g., 10 liters) with magnetic stirrer. Plasma is contacted with low concentration ethanol, e.g. 8-12%, for a period that can be extended to several days and in each case is greater than 24 hours. Then, the supernatant is decanted off which can be used in the preparation of other 15 derivatives.
Man fracentrifugerer bundfaldet, vasker det med ethanol stadig ved lav temperatur, dvs. ved 0-6°C og med 8-12%, fortrinsvis 10% alkohol, hvorefter man igen centrifugerer.The precipitate is centrifuged, it is still washed with ethanol at low temperature, ie. at 0-6 ° C and with 8-12%, preferably 10% alcohol, and then centrifuged again.
20 Nu gensolubiliserer man bundfaldet i en Tris/ci- tratpuffer, hvorefter man fortrinsvis indkoncentrerer, filtrerer og med fordel frysetørrer før videre behandling. Man kan også benytte det direkte til videre forarbejdning, dvs. i ikke frysetørret stand.20 The precipitate is now re-solubilized in a Tris / citrate buffer, and then concentrated, filtered and preferably freeze-dried before further treatment. It can also be used directly for further processing, ie. in freeze-dried condition.
25 Den følgende behandling til opnåelse af et pro teinkoncentrat ifølge opfindelsen kan foregå ifølge to forskellige udførelsesformer.The following treatment for obtaining a protein concentrate according to the invention can be carried out according to two different embodiments.
Ved en første udførelsesform solubiliserer man det frysetørrede produkt eller den tilsvarende friske 30 pasta i vand eller i Tris/citratpuffer, der med fordel indeholder lysin, og man behandler opløsningen med varm fortyndet ethanol.In a first embodiment, the freeze-dried product or the corresponding fresh paste is solubilized in water or in Tris / citrate buffer, which advantageously contains lysine, and the solution is treated with hot diluted ethanol.
35 7 DK 173568 B135 7 DK 173568 B1
Ved denne udførelsesfortn solubiliserer man det frysetørrede fibrinogen eller den tilsvarende friske pasta til et proteinindhold på ca. 50 g/1 og behandler det med 8-12% ethanol ved en temperatur på 30-40eC, 5 foretrukket ved 35eC, og i ca. 1½ time. Dette trin, der fortrinsvis gennemføres under tilstedeværelse af lysln, bidrager til at korrigere solubiliseringen af frysetørret biologisk lim og giver forøget sikkerhed, hvad angår inaktivering af mulige sygdomsfremmende vira, 10 deriblandt AIDS virus.In this embodiment, the freeze-dried fibrinogen or the corresponding fresh paste is solubilized to a protein content of ca. 50 g / l and treat it with 8-12% ethanol at a temperature of 30-40 ° C, preferably at 35 ° C, and for approx. 1½ hours. This step, preferably carried out in the presence of lysine, contributes to correcting the solubilization of freeze-dried biological glue and provides increased security with respect to inactivation of possible disease-promoting viruses, including the AIDS virus.
Mængden af anvendt lysin gør det muligt f.eks. at have en koncentration på 0,1-0,2 g/g protein i det anvendelsesklare produkt.The amount of lysine used makes it possible e.g. having a concentration of 0.1-0.2 g / g protein in the ready-to-use product.
Ethanolen fjernes ved ultrafiltrering eller dia-15 filtrering mod en puffer, der foretrukket indeholder en mængde lysin, der svarer til den ønskede mængde i det brugsklare produkt, men med fordel ikke indeholder citrat, så koncentrationen af denne forbindelse i slutproduktet er så lav som muligt, idet den tilsyneladende 20 påvirker koaguleringen i ugunstig retning. Man kan f.eks. benytte en Tris/NaCl/lysinpuffer.The ethanol is removed by ultrafiltration or di-filtration against a buffer which preferably contains an amount of lysine which corresponds to the desired amount in the ready-to-use product, but preferably does not contain citrate, so that the concentration of this compound in the final product is as low as possible. , the apparent 20 adversely affecting coagulation. One can, for example. use a Tris / NaCl / lysine buffer.
Produktet filtreres derefter under sterile omstændigheder, anbringes i den beholder, det senere skal anvendes fra, og frysetørres.The product is then filtered under sterile conditions, placed in the container from which it will later be used, and freeze-dried.
25 Ifølge den anden udførelsesform behandler man det frysetørrede produkt eller den tilsvarende friske pasta efter solubilisering med et trin til inaktivering af virus, f.eks. ved behandling med opløsningsmiddel og detergent. Remanenserne ved denne behandling elimineres 30 derefter, foretrukket ved udfældelse i kulden under anvendelse af fortyndet ethanol.According to the second embodiment, the lyophilized product or the corresponding fresh paste is treated after solubilization with a step of inactivating virus, e.g. by solvent and detergent treatment. The residues in this treatment are then eliminated, preferably by precipitation in the cold using diluted ethanol.
Ved denne udførelsesform resolubiliserer man det frysetørrede fibrinogen eller den tilsvarende fri- 8 DK 173568 B1 ske pasta til et proteinindhold på ca. 20 g/1. Det underkastes derefter en behandling til inaktivering af virus, f.eks. en behandling med opløsningsmiddel og detergent ifølge opfindelsen.In this embodiment, the freeze-dried fibrinogen or the corresponding free paste is resolubilized to a protein content of approx. 20 g / l. It is then subjected to a treatment for inactivating viruses, e.g. a solvent and detergent treatment according to the invention.
5 Dette trin, der med fordel udføres ved tempera tur, der er højere end 24°C og en tidsperiode, der er længere end 6 timer, giver forøget sikkerhed, hvad angår inaktivering af mulige sygdomsfremkaldende vira, deriblandt AIDS virus og hepatitis virus. Det udføres 10 foretrukket under tilstedeværelse af lysin i en koncentration, der svarer til lysinindholdet i produktet, der er klar til anvendelse, nemlig ca. 0,1-0,2 g/g proteiner .Advantageously, this step, carried out at a temperature higher than 24 ° C and a time period longer than 6 hours, provides increased security with respect to inactivation of possible disease-causing viruses, including AIDS virus and hepatitis virus. It is preferably carried out in the presence of lysine at a concentration corresponding to the lysine content of the ready-to-use product, viz. 0.1-0.2 g / g proteins.
Man opnår eliminering af midlerne til inaktive-15 ring af vira sammen med en voksende renhed af proteinet ved genudfældelse af proteinerne ved lav temperatur under anvendelse af 8-12% ethanol. Efter centrifugering er midlerne til inaktivering af vira og de kontamine-rende proteiner, såsom albumin, i virkeligheden elimi-20 neret i supernatanten. Hvis det er nødvendigt, kan man igen bringe bundfaldet i kontakt med ethanolopløsningen til opnåelse af en bedre eliminering af midlerne til inaktivering af vira.Elimination of the virus-inactivating agents together with a growing purity of the protein is achieved by low precipitation of the proteins at low temperature using 8-12% ethanol. After centrifugation, the agents for inactivating viruses and the contaminating proteins, such as albumin, are in fact eliminated in the supernatant. If necessary, the precipitate can again be contacted with the ethanol solution to obtain a better elimination of the virus inactivation agents.
Bundfaldet solubiliseres igen i en Tris/citrat/ 25 lysinpufferopløsning, hvorefter det ultrafiltreres og diafiltreres før sterilfiltrering og videre fordeling. Formålet med denne ultrafiltrering er at fjerne ethanol og citrat, men også at opretholde et konstant ly-sinindhold på 0,1-0,2 g/g proteiner.The precipitate is solubilized again in a Tris / citrate / 25 lysine buffer solution, after which it is ultrafiltered and diafiltered before sterile filtration and further distribution. The purpose of this ultrafiltration is to remove ethanol and citrate, but also to maintain a constant content of 0.1-0.2 g / g protein.
30 Når koncentratet ifølge opfindelsen skal benyt tes som en biologisk lim, tilvejebringer man genopløsning af limen før anvendelse af produktet ved hjælp af en vandig aprotininopløsning med en styrke på 10000 IE/ml, idet man blander den dannede opløsning med cal-35 ciumaktiveret thrombin med en styrke på 500 JE/ml.When the concentrate according to the invention is to be used as a biological glue, redissolve the glue before using the product by means of an aqueous aprotinin solution of strength 10000 IU / ml, mixing the resulting solution with calcium-activated thrombin with a strength of 500 JE / ml.
Produktet kan enten benyttes på flydende form og påføres ved hjælp af et dobbelt nålesystem (på det ene 9 DK 173568 B1 sted den rekonstruerede biologiske lim og på den andet sted calciumaktiveret thrombin) eller på tør form (som et pulver) eller ved hjælp af et spraysystem.The product can either be used in liquid form and applied by a double needle system (in one place the reconstructed biological glue and in the other place calcium activated thrombin) or in dry form (as a powder) or by means of a spray system.
De følgende eksempler skal illustrere opfindel-5 sen uden at begrænse dens omfang.The following examples are intended to illustrate the invention without limiting its scope.
Eksempel 1 A. Fremstilling af proteinkoncentrat 10Example 1 A. Preparation of Protein Concentrate 10
Det anvendte plasma tilvejebringes ved centrifugering af blod og fryses til -35°C senest 6 timer efter aftapning af blod. Til fremstilling af koncentratet optøes det og opvarmes til 37eC. Derefter under-15 kastes det udfældning med 10% ethanol, pH = 7,2, et proteinindhold på 52 g/1 og en temperatur på 4eC. Blandingen holdes under omrøring i 30 minutter, og man lader den derpå henstå til bundfældelse i mindst 24 timer ved 4°C.The plasma used is obtained by blood centrifugation and frozen to -35 ° C within 6 hours of blood draw. To prepare the concentrate, it is thawed and heated to 37 ° C. It is then subjected to precipitation with 10% ethanol, pH = 7.2, a protein content of 52 g / l and a temperature of 4 ° C. The mixture is kept stirring for 30 minutes and then allowed to settle for at least 24 hours at 4 ° C.
20 Man fracentrifugerer ethanolsupernatanten og op samler bundfaldet, der især er beriget hvad angår fibrinogen og faktor XIII. Bundfaldet vaskes grundigt med en 10% opløsning af ethanol, der i forvejen er afkølet til 4°C og centrifugeres derefter igen. Nu gensolubili-25 seres bundfaldet under anvendelse af en Tris/citratpuf-feropløsning, hvorefter det indkoncentreres til 15-20 g/1 proteiner, filtreres og frysetørres, hvis det er nødvendigt.The ethanol supernatant is centrifuged and the precipitate collected, which is particularly enriched in fibrinogen and factor XIII, is collected. The precipitate is washed thoroughly with a 10% solution of ethanol which has already cooled to 4 ° C and then centrifuged again. Now, the precipitate is re-solubilized using a Tris / citrate buffer solution, then concentrated to 15-20 g / l proteins, filtered and lyophilized if needed.
Det dannede produkt bringes igen i suspension 30 med en koncentration på 50 g/1 proteiner under anvendelse af en 3 g/1 lysinopløsning (svarende til et ly-sinindhold i slutproduktet på 0,1-0,2 g/g proteiner), hvorefter det for anden gang behandles med 10% ethanol i 1½ time ved 35°C.The resulting product is brought back into suspension 30 at a concentration of 50 g / l proteins using a 3 g / l lysine solution (corresponding to a lysine content in the final product of 0.1-0.2 g / g proteins), after which it is treated for the second time with 10% ethanol for 1½ hours at 35 ° C.
35 Efter diafiltrering til fjernelse af citrat og ethanol og tilpasning af proteinindholdet til den øn- 10 DK 173568 B1 skede værdi, f.eks. 35 g/1, filtrerer man koncentratet, anbringer det under sterile omstændigheder i en beholder og frysetørrer det her til senere brug som f.eks. ved 0,5, 1, 2 eller 5 ml opløsning.After diafiltration to remove citrate and ethanol and adjust the protein content to the desired value, e.g. 35 g / l, filter the concentrate, place it under sterile conditions in a container and freeze-dry it here for later use, e.g. at 0.5, 1, 2 or 5 ml of solution.
5 B. Biokemisk analyse af proteinkoncentratet5 B. Biochemical analysis of the protein concentrate
Proteinsammensætningen af produktet er som følger (udtrykt pr. gram proteiner): 10 Fibrinogen 0,70-0,75 gThe protein composition of the product is as follows (expressed per gram of protein): 10 Fibrinogen 0.70-0.75 g
Endogen faktor XIII 100-250 IEEndogenous factor XIII 100-250 IU
Fibronectin 0,05-0,10 gFibronectin 0.05-0.10 g
Eksempel 2 15 A. Fremstilling af proteinkoncentratExample 2 A. Preparation of protein concentrate
Det anvendte plasma opnås ved centrifugering af blod og fryses til -35°C inden 6 timer efter aftapnin-20 gen. Til fremstilling af koncentratet optøes det og opvarmes til 37°C. Derefter behandles det med udfældelse med en 10% ethanol med en pH-værdi på 7,2, ved et proteinindhold på 52 g/1 og en temperatur på 4°C. Blandingen holdes under omrøring i 30 minutter og henstår så 25 til bundfældning i mindst 24 timer ved 4°C.The plasma used is obtained by centrifugation of blood and frozen to -35 ° C within 6 hours of collection. To prepare the concentrate, it is thawed and heated to 37 ° C. Then it is treated with precipitation with a 10% ethanol with a pH of 7.2, at a protein content of 52 g / l and a temperature of 4 ° C. The mixture is kept under stirring for 30 minutes and then allowed to settle for at least 24 hours at 4 ° C.
Man fjernes ethanolsupernatanten ved centrifugering og indsamler bundfaldet, der er specielt beriget, hvad angår fibrinogen og faktor XIII. Bundfaldet vaskes grundigt med en 10% opløsning af ethanol, der i 30 forvejen er afkølet til 4°c, hvorefter det igen centrifugeres .The ethanol supernatant is removed by centrifugation and the precipitate which is specially enriched for fibrinogen and factor XIII is collected. The precipitate is washed thoroughly with a 10% solution of ethanol which has been pre-cooled to 4 ° C for 30 minutes and then centrifuged again.
Nu genopløses bundfaldet under anvendelse af en Tris/citratpufferopløsning, hvorefter det indkoncentre-res til 15-20 g/1 proteiner, filtreres og frysetørres, 35 hvis det kræves.Now, the precipitate is redissolved using a Tris / citrate buffer solution, then concentrated to 15-20 g / l of proteins, filtered and lyophilized if required.
Det opnåede produkt udsuspenderes igen til en koncentration på ca. 20 g/1 proteiner under anvendelse 11 DK 173568 B1 af en lysinopløsning, der svarer til lysinindholdet i slutproduktet på 0,1-0,2 g/g proteiner, hvorefter det behandles med opløsningsmiddel og detergent, nemlig 0,3% TNBP og 1% Tween 80 i en tid, der overstiger 6 ti-5 mer og en temperatur, der er højere end 24°C.The product obtained is again suspended to a concentration of approx. 20 g / l of proteins using 11 lysine solution corresponding to the lysine content of the final product of 0.1-0.2 g / g of proteins, after which it is treated with solvent and detergent, namely 0.3% TNBP and 1 % Tween 80 for a time exceeding 6 hours and a temperature higher than 24 ° C.
Opløsningen behandles derefter med alkoholud-fældelse under anvendelse af 10% ethanol ved 4°c og henstår til bundfældning i ca. 10 timer, hvorefter den dekanteres.The solution is then treated with alcohol precipitation using 10% ethanol at 4 ° C and allowed to settle for approx. 10 hours, then decant.
10 Efter centrifugering og fjernelse af superna- tanten (der kan indeholde midler til inaktivering af vira) solubiliserer man bundfaldet under anvendelse af en Tris/citratpuffer med indhold af lysin i en mængde, der svarer til 0,1-0,2 g lysin pr. gram proteiner.After centrifugation and removal of the supernatant (which may contain virus inactivation agents), the precipitate is solubilized using a Tris / citrate buffer containing lysine in an amount equal to 0.1-0.2 g lysine per . grams of proteins.
15 Efter diafiltrering til tilpasning af ionstyr ken, fjernelse af citrat og ethanol (men ikke lysin) og tilpasning af proteinindholdet til en passende værdi, f.eks. ca. 35 g/1, filtrerer man koncentratet, anbringer det under sterile omstændigheder i den beholder, 20 det senere skal benyttes i som 0,5, 1, 2 eller 5 ml opløsning, og frysetørrer det.After diafiltration to adjust the ionic strength, remove citrate and ethanol (but not lysine) and adjust the protein content to an appropriate value, e.g. ca. 35 g / l, the concentrate is filtered, placed under sterile conditions in the container, 20 to be subsequently used as 0.5, 1, 2 or 5 ml of solution and freeze-dried.
B. Biokemisk analyse af proteinkoncentratet 25 Proteinsammensætningen af produktet er som føl ger (udtrykt pr. gram proteiner):B. Biochemical Analysis of the Protein Concentrate 25 The protein composition of the product is as follows (expressed per gram of protein):
Fibrinogen 0,90-0,95 gFibrinogen 0.90-0.95 g
Endogen faktor XIII 150-300 IEEndogenous factor XIII 150-300 IU
Fibronectin 0,03-0,06 g 30 På tegningen vises resultaterne af elektrofore- seanalyse, henholdsvis for dette koncentrat (kurve la) og for kommercielle koncentrater til anvendelse som biologisk lim (kurverne lb-ld).Fibronectin 0.03-0.06 g 30 The drawing shows the results of electrophoresis analysis, respectively for this concentrate (curve 1a) and for commercial concentrates for use as biological adhesive (curves 1b-1d).
Der ses en betragtelig større renhed af kompo-35 nenter i zone y (fibrinogen) af produktet ifølge opfindelsen ved disse kurver.Significantly greater purity of components in zone γ (fibrinogen) of the product of the invention is seen by these curves.
12 DK 173568 B112 DK 173568 B1
Som nævnt ovenfor har koncentratet ifølge opfindelsen udmærkede solubiliserings- og stabilitetsegenskaber .As mentioned above, the concentrate according to the invention has excellent solubilization and stability properties.
Koncentratet kan i praksis solubiliseres på 5 mindre end 10 minutter, i virkeligheden mindre end 5 minutter, ikke blot ved 37°C, men også ved 20°C uden et specielt apparat, blot ved hjælp af almindelige roterende håndbevægelser.In practice, the concentrate can be solubilized in 5 less than 10 minutes, in fact less than 5 minutes, not only at 37 ° C, but also at 20 ° C without a special apparatus, simply by ordinary rotary hand movements.
Man observerer ydermere ingen destabilisering af 10 det genfremstillede produkt, der i 24 timer er opbevaret ved 24 eller 37°C.Furthermore, no destabilization of the reconstituted product stored for 24 hours at 24 or 37 ° C is observed.
Hvad angår den biologiske lim ifølge opfindelsen kan det fastslås, at den har udmærkede egenskaber til en sådan anvendelse. Dens klæbeevne er i virkeligheden 15 større end 100 g/cm^ ifølge en afprøvning med klæbning af hudstriber på dyr, en værdi der er større end hvad man opnår med andre lim fremstilles fra udfrysnings-produkter. Denne klæbeevne opretholdes på til 90% efter gendannelsen af den biologiske lim og opbevaring i 24 20 timer ved 4°C eller ved 20°C. Derudover ser man ingen udskillelser ved sammenblanding af proteinkoncentratet og calciumaktiveret thrombin. Kliniske afprøvninger har vist, at denne lim i høj grad opfylder de nødvendige krav: hurtig solubilisering, stabilitet ved anvendel- 25 sestemperaturer, mulighed for forsinket anvendelse.As to the biological adhesive of the invention, it can be stated that it has excellent properties for such use. Its adhesive is in fact 15 greater than 100 g / cm 2 according to a test with adhesive skin streaks to animals, a value greater than what is obtained with other adhesives produced from freezing products. This adhesive is maintained at up to 90% after the recovery of the biological adhesive and storage for 24 hours at 4 ° C or at 20 ° C. In addition, no secretions are seen when mixing the protein concentrate and calcium-activated thrombin. Clinical trials have shown that this adhesive largely meets the required requirements: rapid solubilization, stability at operating temperatures, possibility of delayed use.
Anvendelsen af limen ifølge opfindelsen følger af dens egenskaber: dens klæbende og blodstandsende egenskaber gør den til et værdifuldt supplement ved kirurgiske operationer, og man opnår herved en kortere 30 operationstid. Derudover gør dens bakteriostatiske egenskaber det muligt at opnå en hurtigere og bedre heling af sår og suturer.The use of the adhesive according to the invention is due to its properties: its adhesive and anti-corrosive properties make it a valuable adjunct to surgical operations, thereby achieving a shorter operating time. In addition, its bacteriostatic properties enable faster and better healing of wounds and sutures.
Den kan således anvendes ved mange kirurgiske indgreb: 35 Plastisk kirurgi og mikrokirurgi: hudtransplan tationer til brandsår, befæstning af hudstrimler, fjernelse af hud, operationer i øjenlågene.It can thus be used in many surgical procedures: 35 Plastic surgery and microsurgery: skin transplants for burns, skin strips, skin removal, eyelid surgery.
13 DK 173568 B113 DK 173568 B1
Neurokirurgi: plastisk kirurgi og hjernehindesuturer, standsning af blod ved fjernelse af svulster, standsning af blod ved karoperationer ved hjernen.Neurosurgery: plastic surgery and cerebral sutures, stopping blood from tumors, stopping blood from blood vessels at the brain.
Cardiovaskulær kirurgi: befæstning af suturer 5 ved indsætning af kunstige blodkar, ved operationer i aorta og ved aneurismer.Cardiovascular surgery: attachment of sutures 5 by insertion of artificial blood vessels, by operations in aorta and by aneurysms.
Almen og abdominal kirurgi: sammensyninger ved underlivsorganerne (milt, nyre, lever......) eller le- verbiopsi, digestiv anastomosis, fistler, standsning af 10 blod under operationer.General and abdominal surgery: joints of the abdominal organs (spleen, kidney, liver ......) or liver biopsy, digestive anastomosis, fistulas, quitting of blood during surgery.
Knoglekirurgi: heling af knogleoverflade, sammensyning af sener, heling af knoglebetændelse.Bone surgery: healing of bone surface, constriction of tendons, healing of bone inflammation.
Stomatologi: standsning af blod ved tandudtrækning hos patienter med høj blødningsrisiko (blødere).Stomatology: stopping of blood upon extraction in patients with high bleeding risk (bleeding).
15 Otorhinolaryngologi: heling af sår fra mellem ørebetændelse, fjernelse af mandler.15 Otorhinolaryngology: healing of wounds from between ear inflammation, removal of tonsils.
Limen ifølge opfindelsen kan fremstilles ud fra humant eller dyrisk plasma og kan således med fordel benyttes til behandling af mennesker og dyr.The adhesive according to the invention can be prepared from human or animal plasma and can thus be advantageously used for the treatment of humans and animals.
20 Hvad angår injicerbart fibrinogen ifølge opfin delsen ses det, at det også har udmærkede anvendelsesegenskaber, og at dets afbalancerede sammensætning gør det til et værdifuldt terapeutisk middel til behandling af hypofibronogenese eller afibrinogenese eller af syn-25 dromet med akut defibrinering.In the case of injectable fibrinogen according to the invention, it is seen that it also has excellent application properties and that its balanced composition makes it a valuable therapeutic agent for the treatment of hypofibronogenesis or afibrinogenesis or of the syndrome of acute defibrination.
Til behandling af medfødte mangler er den sædvanlige dosis 1-4 g, afhængig af patienten vægt, idet man tager i betragtning, at halveringstiden for fibrinogen er 3-4 dage, og at indholdet i plasma til opnåel-30 se af en tilfredsstillende koagulering af ca. 1 g/1.For the treatment of congenital deficiencies, the usual dose is 1-4 g, depending on the patient's weight, taking into account that the half-life of fibrinogen is 3-4 days and that the plasma content to achieve a satisfactory coagulation of ca. 1 g / l.
ved tilfælde med akut defibrinering kan dosis variere fra 2-10 g afhængig af omstændighederne. De terapeutiske produkter ifølge opfindelsen kan fremstilles ud fra humant eller dyrisk plasma, og således ikke blot 35 anvendes på mennesker, men også i veterinær terapi.in cases of acute defibrination the dose may vary from 2-10 g depending on the circumstances. The therapeutic products of the invention can be prepared from human or animal plasma and thus not only used in humans but also in veterinary therapy.
Claims (8)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8710798 | 1987-07-30 | ||
| FR8710798A FR2618784B1 (en) | 1987-07-30 | 1987-07-30 | CONCENTRATE OF THROMBIN-COAGULABLE PROTEINS, PROCESS FOR OBTAINING SAME AND ITS USE AS A BIOLOGICAL GLUE |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK424188D0 DK424188D0 (en) | 1988-07-29 |
| DK424188A DK424188A (en) | 1989-01-31 |
| DK173568B1 true DK173568B1 (en) | 2001-03-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| DK198804241A DK173568B1 (en) | 1987-07-30 | 1988-07-29 | Process for Preparing a Plasma-Based, Thrombin Coagulable Protein Concentrate and Protein Concentrate |
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| EP (1) | EP0305243B1 (en) |
| JP (1) | JP2787317B2 (en) |
| AT (1) | ATE73341T1 (en) |
| CA (1) | CA1341376C (en) |
| DE (1) | DE3869018D1 (en) |
| DK (1) | DK173568B1 (en) |
| ES (1) | ES2039666T3 (en) |
| FR (1) | FR2618784B1 (en) |
| GR (1) | GR3004047T3 (en) |
| NO (1) | NO174929C (en) |
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| JPH0431072Y2 (en) * | 1988-03-14 | 1992-07-27 | ||
| FR2639958B1 (en) * | 1988-12-06 | 1992-08-21 | Lille Transfusion Sanguine | BIOLOGICAL SUPPORT FOR CELL CULTURES CONSISTING OF PLASMATIC PROTEINS COAGULATED BY THROMBIN, ITS USE FOR THE CULTURE OF KERATINOCYTES, THEIR RECOVERY AND THEIR TRANSPORT FOR THERAPEUTIC USE |
| US6197325B1 (en) | 1990-11-27 | 2001-03-06 | The American National Red Cross | Supplemented and unsupplemented tissue sealants, methods of their production and use |
| US6117425A (en) | 1990-11-27 | 2000-09-12 | The American National Red Cross | Supplemented and unsupplemented tissue sealants, method of their production and use |
| US6054122A (en) | 1990-11-27 | 2000-04-25 | The American National Red Cross | Supplemented and unsupplemented tissue sealants, methods of their production and use |
| US7208179B1 (en) | 1990-11-27 | 2007-04-24 | The American National Red Cross | Methods for treating disease and forming a supplemented fibrin matrix |
| DE4202667C1 (en) | 1992-01-29 | 1993-05-13 | Behringwerke Ag, 3550 Marburg, De | |
| AU4693093A (en) * | 1992-07-27 | 1994-02-14 | Haemacure Biotech Inc. | Biocompatible surgical implant |
| FR2696095B1 (en) * | 1992-09-30 | 1994-11-25 | Inoteb | Biological glue based on proteins coagulable by thrombin, enriched in platelet factors, its preparation and its application. |
| US5290918A (en) * | 1993-02-23 | 1994-03-01 | Haemacure Biotech Inc. | Process for the obtention of a biological adhesive made of concentrated coagulation factors by acidic precipitation |
| US5395923A (en) * | 1993-02-23 | 1995-03-07 | Haemacure-Biotech, Inc. | Process for the obtention of a biological adhesive made of concentrated coagulation factors by "salting-out" |
| DK0835667T3 (en) * | 1996-03-15 | 2005-12-05 | Chemo Sero Therapeut Res Inst | Tissue adhesive suitable for spray application |
| WO1998055140A1 (en) * | 1997-06-05 | 1998-12-10 | Omrix Biopharmaceuticals S.A. | Fibrinogen concentrate from human plasma |
| GB0216001D0 (en) | 2002-07-10 | 2002-08-21 | Nat Blood Authority | Process and composition |
| US20060019234A1 (en) * | 2004-07-22 | 2006-01-26 | Shanbrom Technologies, Llc | Modern blood banking employing improved cell preservation composition |
| FR2887883B1 (en) * | 2005-06-29 | 2007-08-31 | Lab Francais Du Fractionnement | PROCESS FOR SEPARATING FIBRINOGEN PROTEINS, FACTOR XIII AND BIOLOGICAL GLUE OF SOLUBILIZED PLASMA FRONTION AND PREPARATION OF LYOPHILIZED CONCENTRATES OF SAID PROTEINS |
| FR2894831B1 (en) | 2005-12-16 | 2008-02-15 | Lab Francais Du Fractionnement | THROMBIN FREE BIOLOGICAL GLUE AND USE THEREOF AS MEDICAMENT. |
| FR2952641B1 (en) | 2009-11-18 | 2012-01-13 | Lfb Biomedicaments | PROCESS FOR TREATING BLOOD PLASMA COMPRISING A DISPERSION WASH STEP |
| FR3032621A1 (en) | 2015-02-13 | 2016-08-19 | Lab Francais Du Fractionnement | BIOLOGICAL GLUE AND ITS USE AS MEDICINE |
| US20180169297A1 (en) | 2015-06-12 | 2018-06-21 | Laboratoire Français Du Fractionnement Et Des Biotechnologies | Injectable composition of factor vii and fillers |
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| US4156681A (en) * | 1974-03-28 | 1979-05-29 | Plasmesco Ag | Process for isolating albumin from blood |
| AT359653B (en) * | 1979-02-15 | 1980-11-25 | Immuno Ag | METHOD FOR PRODUCING A TISSUE ADHESIVE |
| AT359652B (en) * | 1979-02-15 | 1980-11-25 | Immuno Ag | METHOD FOR PRODUCING A TISSUE ADHESIVE |
| EP0068047B1 (en) * | 1981-06-25 | 1986-07-23 | Serapharm GmbH & Co. KG | Enriched plasma derivative for promoting wound sealing and wound healing |
| US4374061A (en) * | 1981-07-27 | 1983-02-15 | Center For Blood Research, Inc. | Means and methods for purifying Clq, Clr and Cls |
| DE3203775A1 (en) * | 1982-02-04 | 1983-08-11 | Behringwerke Ag, 3550 Marburg | FIBRINOGEN PREPARATION, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| DE3228502A1 (en) * | 1982-07-30 | 1984-02-02 | Behringwerke Ag, 3550 Marburg | METHOD FOR PRODUCING THE C1 INACTIVATOR AND ITS USE |
| US4540573A (en) * | 1983-07-14 | 1985-09-10 | New York Blood Center, Inc. | Undenatured virus-free biologically active protein derivatives |
| AT389815B (en) * | 1984-03-09 | 1990-02-12 | Immuno Ag | METHOD FOR INACTIVATING VARIABLE FILTERABLE DISEASERS IN BLOOD PRODUCTS |
| US4613501A (en) * | 1984-12-21 | 1986-09-23 | New York Blood Center, Inc. | Inactivation of viruses in labile blood derivatives |
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1987
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1988
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- 1988-07-28 ES ES198888401961T patent/ES2039666T3/en not_active Expired - Lifetime
- 1988-07-28 AT AT88401961T patent/ATE73341T1/en not_active IP Right Cessation
- 1988-07-28 EP EP88401961A patent/EP0305243B1/en not_active Expired - Lifetime
- 1988-07-28 DE DE8888401961T patent/DE3869018D1/en not_active Expired - Lifetime
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- 1988-07-29 JP JP63191977A patent/JP2787317B2/en not_active Expired - Lifetime
- 1988-07-29 DK DK198804241A patent/DK173568B1/en not_active IP Right Cessation
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1992
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| NO883342D0 (en) | 1988-07-28 |
| NO883342L (en) | 1989-01-31 |
| GR3004047T3 (en) | 1993-03-31 |
| ATE73341T1 (en) | 1992-03-15 |
| EP0305243B1 (en) | 1992-03-11 |
| FR2618784B1 (en) | 1990-04-06 |
| CA1341376C (en) | 2002-07-16 |
| DE3869018D1 (en) | 1992-04-16 |
| ES2039666T3 (en) | 1993-10-01 |
| NO174929B (en) | 1994-04-25 |
| NO174929C (en) | 1994-08-03 |
| FR2618784A1 (en) | 1989-02-03 |
| DK424188D0 (en) | 1988-07-29 |
| EP0305243A1 (en) | 1989-03-01 |
| DK424188A (en) | 1989-01-31 |
| JP2787317B2 (en) | 1998-08-13 |
| JPH02114A (en) | 1990-01-05 |
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